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STUDY OF GENETIC DIVERSITY OF TOMATO

VARIETIES AND VALIDATION OF DISEASE


RESISTANT MARKERS

Department of Biotechnology
HOD : Dr S M Gopinath
Project guide : Dr Ismail Shareef M
External guide: Dr Devaraj Achar

Anagha S K (1AY13BT001)
Bhavani R (1AY13BT005)
Harusha S (1AY13BT011)
Content
Introduction.
Objective.
Collection and sowing of experimental material.
DNA Isolation and Extraction.
Genotyping of tomato samples using specific
markers.
Standardization of PCR amplification.
Agarose Gel Electrophoresis.
Scoring of Genotyping data.
Introduction
Tomato, Solanum lycopersicum L., is a member of the Solanaceae
family grown extensively in central, southern and southeast Asia and
in a number of African countries. It is a good source of minerals and
vitamins, like other prominent Solanaceous vegetables such as
Eggplant, potato, pepper and hence, it is important for human
nutrition. The consumption of fruits and vegetables is important for
the prevention of several diseases, and there is growing interest
among consumers in the health benefits of foods. Several studies
report approaches to minimize losses and maintain nutritional value
in fruits and vegetables.
The analysis of genotypes derived from different geographical areas is
important to study genetic diversity. In order to assess genetic diversity
as well as to discriminate tomato cultivars and related Solanum
lycopersicum species, morphological and biochemical approaches are
used. On the other hand, molecular markers have enormous potential
to explore genetic diversity by detecting polymorphisms. They are
useful tools for breeding, genotype identification, and the
determination of genome organization and evolution in plants. Few
studies have been performed to determine the genetic diversity of
tomato using random amplified polymorphic DNA (RAPD), AFLP, simple
sequence repeats (SSR). A comparative genetic linkage map based on
tomato cDNA, genomic DNA, and expressed sequence tag (EST)
markers was constructed for tomato.
On the other hand, RAPD markers provide a rapid, inexpensive and
effective system for studying plant genetic relationships. They are
particularly suitable for less well-known species because they can be
applied without prior knowledge of DNA sequence information
Objective
Isolation of DNA from diverse eggplant varieties
Standardization of PCR Amplification
Molecular profiling of eggplant varieties by RAPD
markers
Principal component analysis (PCA) of
morphological traits.
Detection of genetic polymorphisms and
construction of dendrogram.
Collection and sowing of experimental
material
1. 26 different varieties of tomato samples were
collected and these are named as TP-1 to TP-26.
2. The seeds of these 26 samples were isolated and
sterilized thoroughly.
3. These seeds were placed on 26 different
germinating sheet for a week to obtain plantlet.
4. After obtaining the plantlet, it is used for DNA
isolation.
DNA Isolation and Extraction.

Grind 30mg of sample in 500l of extraction buffer

Transfer to eppendrof tubes. Incubate at 60C for 25 minutes

Cool to room temperature. Add equal volume of 24:1


chloroform:isoamyl alcohol. Keep tubes on shaker for 15minutes.
Centrifuge at 9000rpm for 12minutes at room
temperature.

Transfer supernatant to fresh tubes. At equal


volume of ice cold isopropanol + 0.1V of 5M NaCl.

Gently mix and incubate at -20C for 1-2hrs.


Centrifuge at 9000rpm for 10minutes at 4C.

Decant supernatant. To the pellet add 100l of 70%


ethanol. Centrifuge at 9000rpm foe 10minutes.

Decant ethanol, air dry pellet and suspend in 50l


of 1X TE buffer.
Genotyping of tomato samples using
specific markers
Genotyping is done to detect variation in gene, which
codes for particular disease and validate whether the
sample is resistant or susceptible according to their
genetic makeup.
They are various markers used to detect gene line.
Commonly used markers for genome of tomato
samples include SCAR, CAPS, and SSR.
a. SCAR(Sequenced Characterized Amplified Region)
SCARs are locus specific and have been applied in
gene mapping studies and marker assisted selection. It is
used when there is variation in the length of a gene.
b. CAPS(Cleaved Amplified Polymorphic Sequence)
CAPS marker have been applied predominantly in
gene mapping studies. The limited size of the amplified
fragments should range between 300-1800bp. It is used
when there is mutation in the gene having same length.

c. SSR(Simple Sequence Repeats)


SSR marker plays an important role in understanding
the genetic diversity among the species and within inbred
lines at the molecular level. It is used when there is
multiple repeats of same sequence in a gene.
Standardization of PCR amplification.
Gene Primer Type Ta REN Disease R S

BW SLM 6-17 SSR 55 - 186bp </>

BW SLM 6-94 SSR 58 - 276bp </>

BW SLM 6-110 SSR 55 - 274bp </>

BW SLM 6-118 SSR 55 - 183bp </>

BW SLM 6-119 SSR 55 - 255bp </>

BW SLM 6-124 SSR 55 - 292bp </>

BW SLM 6-136 SSR 55 - 290bp </>

BW SLM 12-2 SSR 55 - 240bp </>

BW SLM 12-10 SSR 55 - 210bp </>


Genotyping Data
Gene Primer Type Ta REN Disease R S

TY1 TG97 CAPS 55 Tag I TYLCV 303+95bp 398bp

TY2 T0302 SCAR 55 NA Begomovirus 900bp 800bp

TY3 P6-25 SCAR 55 NA TYLCV and 450/630/660 320bp


ToMoV bp
TY4 C2AT5g63460 CAPS 55 Hinf-I TYLCV(15.7%) 850bp 650bp

TY5 SinacI CAPS 55 Taq-I 410bp 370bp

Mi Mi-23 SCAR 55 NA Root-knot 380bp 430bp


nematode

TM2 NCTM19 CAPS 55 Hac-III Tropomyosin 600+277bp 870bp


fruit fly
VE Ve2.3 CAPS 55 Hinc-II Verticillium 610+428bp 1029bp
fungal
PH2 dTG63 CAPS 55 Hinf-I GR/HPR 250bp 220bp
deficiency
Gene Primer Type Ta REN Disease R S
PH3 TG328 CAPS 55 BStN-I Late blight 250bp 500bp
LV CT121 CAPS 55 Hac-III Leveillula 230bp 180bp
Taurica fungus
I2 I2 CAPS 62 Ras-I Fusarium wilt 500bp 230bp
F3 CAPS 57 Hinf-I 800bp 500+220bp
Bspect PTO CAPS 57 Rsa-I 552bp 439+113bp
Bspot RX3 CAPS 60 BSrB-I 275+48 323bp
bp
Tospo SW5 CAPS 50 500bp 500+450bp
BW SLM 6-124 55
BW SLM 6-136 55
BW SLM 6-17 55
BW SLM 6-110 55
BW SLM 6-94 55
BW SLM 12-10 55
BW SLM 12-2 55
TYLCV- Tomato Yellow Leaf Curl Virus
ToMoV- Tomato Mottle Virus
Begomoviru- Major threats to tomato production
Verticillium, Leveillula Taurica is a common fungal
disease causing severe yield and quality losses.
PH-2 caused by deficiency of enzyme glyoxylate
reductase/hydroxypyruvate reductase
Late blight caused by Alternaria solani, a fungal disease
leading to browning in tomato
Fusarium wilt- shrinkage of fruits and leaves by excess
loss of water