Project on Assessment of Genetic Diversity in Wheat Genotypes by using ISSR Molecular Marker.

1. Assessment of Genetic Diversity In Wheat Genotypes By Using
ISSR Molecular Marker
BY
Name: Asif Shaukat Shaikh
Reg. No.: PES/2013/25
Course No.: HOT-481
B.Sc. Agricultural Biotechnology
Modern College of Agricultural Biotechnology
Kule-Dakhane, Paud, Pune.
Guided By
Mr. S.D. Tidke
Assistant Professor
Modern College of Agricultural Biotechnology
Kule-Dakhane, Paud, Pune. 1

2. CONTENTS
 Introduction
 Objectives
 Materials and Methods
 Results and Discussion
 Summary and Conclusion
 Future Prospects
 References
2

3.  Wheat-
Scientific classification:
Triticum aestivum
Kingdom : Plantae
Division : Magnoliophyta
Class : Liliopsida
Order : Poales
Family : Poaceae
Genus : Triticum
3
Fig No.1: Wheat plant
(Choudhary et al., 2016)
introduction

4.  Wheat is the most important cereal crop; it is a stable diet for the
world population and which is a foundation of human nutrition
and of enormous economic importance in worldwide.
(Haleem et al., 2009)
 Wheat possesses the prime importance worldwide amongst all
cereal crops due to the dependence of huge population for
consumption, area under cultivation, production and trading in
international market.
(Chaudhary et al., 2016)
4

5.  The protein contents of wheat grains may vary between 10%-18%
of the total dry matter.
 Gluten protein contents, high molecular weight glutenins 11% and
low molecular weight glutenins 22%.
(Scherf et al., 2016)
 The amount of starch contained in a wheat grain may vary
between 60% and 75% of the total dry weight of the grains.
 Whole grains are good sources of insoluble fiber.
(Sramkova et al., 2009)
5

6.  According to Food Agricultural Organization (FAO) total wheat
utilization in the world is now projected at 736.5 million tonnes, up
22 million tonnes (3.1 %) from 2015-16.
(www.fao.org)
 India is today the second largest wheat producer in the whole
world.
 India has exported 618020.01 MT of wheat to the world for the
worth of Rs. 978.59 crores during the year of 2015-16.
 Major wheat growing states in India are Uttar Pradesh, Punjab,
Haryana, Madhya Pradesh, Rajasthan, Bihar and Gujarat.
(APEDA-2015-16)
6

7.  ISSR are one of the PCR-based markers that have become widely
used in various areas of plant research.
(Deif et al., 2013)
 ISSR are segments of DNA that are flanked at both ends by such a
microsatellite sequences.
 ISSR markers are also popularly known as random amplified
microsatellites (RAMs).
(Ng and Tan, 2015)
7
 Inter Simple Sequence Repeats-

8.  ISSR have a simple procedure, low-cost, good stability and high
reproducibility.
(Razmjoo et al., 2015)
 ISSR have high reproducibility possibly due to the use of longer
primers (16–25 nucleotides) as compared to RAPD primers (10-12
nucleotides).
(Reddy et al., 2002)
 ISSR marker have been successfully used in diversity analysis of
wheat germplasm.
(Goyal et al., 2015)
8

9. OBJECTIVES
 To extract, purify and quantify genomic DNA.
 To study PCR amplification with UBC primers.
 To study ISSR data analysis.
 To construct dendrogram for study genetic diversity.
9

10. MATERIALS AND METHODs
 Collection of seed samples: The seed samples were collected from
National Agricultural Research Station (NARP), Washim.
10
 Materials -
Sr.
No.
Genotypes
Aestivum/
Durum
Released/ In
Pipeline
Sr.
No.
Genotypes
Aestivum/
Durum
Released/ In
Pipeline
1.
Ajantha (MAU) T. aestivum Released
12.
WSM-163A T. aestivum In Pipeline
2.
NIAW-1415 T. aestivum Released
13.
WSM-1757 T. aestivum In Pipeline
3.
NI-5439 T. aestivum Released
14.
WSM-1472 T. aestivum In Pipeline
4.
PKV (Washim) T. aestivum Released
15.
WSM-109 T. aestivum In Pipeline
5.
AKW-3722 T. aestivum Released
16.
WSM-163 T. aestivum In Pipeline
6.
AKW-107 T. aestivum Released
17.
WSM-105 T. aestivum In Pipeline
7.
MACS-1967 T. Durum Released
18.
WSM-42 T. aestivum In Pipeline
8.
LOK-1 T. aestivum Released
19.
WSM-55D T. aestivum In Pipeline
9.
N-59 T. Durum Released
20.
WSM-135 T. aestivum In Pipeline
10.
HD-2189 T. aestivum Released
21.
WSM-154A T. aestivum In Pipeline
11.
HD-2781 T. aestivum Released
22.
WSM-174 T. aestivum In Pipeline
Table No. 1: Description of 22 wheat genotypes

11.  Selection of ISSR primers:
A total of 15 UBC primers for ISSR purchased from
BioResource, Pune.
11
Sr. No. Primer Name Accession Number Primer sequence (5’-3’)
1 UBC-807 217464224 AGA GAG AGA GAG AGA GT
2 UBC-808 217467971 AGA GAG AGA GAG AGA GC
3 UBC-809 217464226 AGA GAG AGA GAG AGA GG
4 UBC-811 217464227 GAG AGA GAG AGA GAG AC
5 UBC-812 217464228 GAG AGA GAG AGA GAG AA
6 UBC-813 217467972 CTC TCT CTC TCT CTC TT
7 UBC-816 217464230 CAC ACA CAC ACA CAC AT
8 UBC-823 217464231 TCT CTC TCT CTC TCT CC
9 UBC-830 217464232 TGT GTG TGT GTG TGT GC
10 UBC-831 217464233 ATA TAT ATA TAT ATA TYA
11 UBC-837 217464234 TAT ATA TAT ATA TAT ART
12 UBC-849 217464235 GTG TGT GTG TGT GTG TYA
13 UBC-857 217464236 ACA CAC ACA CAC ACA CYG
14 UBC-876 217464237 GAT AGA TAG ACA GAC A
15 UBC-880 217464238 GGA GAG GAG AGG AGA
Table No. 2: Details of primers used

12.  Collection of Master mix:
Ready made 2X master mix was collected from BioResource,
Pune.
 Reagents:
• Extraction buffer (CTAB)
• TE buffer
• 50X TAE buffer
• 1X TAE buffer
• Ethidium Bromide (EtBr)
12
Fig No. 2: All genotypes were planted in
field
Sr. No. Components Concentrations
1 PCR buffer 10X
2 dNTPs 10mM
3 Taq DNA Polymerse 3 U
4 MgCl2 2mM
Table No.3: Components and concentrations of 2X master mix

13.  Chemicals:
• Agarose powder
• Bromo phenol blue
• Chloroform
• Ethanol
• Isoamyl alcohol
• Isopropanol
• Proteinase-K
• RNase-H
 Glasswares:
• Beaker
• Conical Flask
• Glass pipette
• Measuring cylinder
• Petri plate
 Instruments:
• Autoclave (Bio-Techniques India)
• Centrifuge machine (Remi)
• Electrophoresis Unit (BioEra)
• Gel documentation machine (Unitech)
• Gradient PCR (Eppendorf)
• Nano Spectrophotometer (BioEra)
• pH meter (EI)
• Refrigerator (Remi)
• U.V. Transilluminator (BioEra)
• Vortex (Remi)
• Water Bath (i-therm)
• Weighing balance (Citizen)
13

14. Overview of Project
14

15. 15
Supernatant was taken in new vial & equal volume chilled isopropanol
was added
Centrifuged at 10,000 rpm for 10 min at 4˚C
Supernatant was taken in new vial & equal volume of chloroform:
isoamyl alcohol (24:1) was added
Centrifuged at 10,000 rpm for 10 min at 4˚C
Crushed leaves were taken in centrifugation tube with 1ml of CTAB
buffer
2 gm of leaves were collected
 Extraction of DNA from wheat leaves:
 Methods-
Contd...

16. 16
Samples were stored at -20°C
Pellets were air-dried at 37˚C and dissolved in 200 µl of TE buffer
DNA pellets washed with 70% ethanol 2-3 times
Centrifuged at 10000 rpm for 10 min at 4°C and supernatant was
discarded
Samples were mixed and incubated at -20°C for 30 min.
Fig No. 4: Procedure for Extraction of DNA
(Doyle and Doyle, 1990)

17.  Agarose Gel Electrophoresis of extracted DNA-
(Ghosh, et al., 2014)
17
Power was switched off when gel was run till 3/4
th
Samples were run at 50 V using 1X TAE buffer for 45 min
Samples were loaded in wells
1X TAE was poured in the gel tank
3 to 5 µl of EtBr was added
Comb was placed in gel tank
Agarose solution clear dissolves completely
Weighed 0.8%of agarose & 1X TAE was added
Fig No. 5: Procedure for Agarose gel electrophoresis

18.  DNA Purification-
 DNA Quantification-
• By U. V. Spectrophotometer:
Add 10 µl of each DNA sample to 900µl TE.
OD260
DNA (µg/ml) = -------------------------
OD280
(Gaikwad, 2015)
18
Dissolved in 500 µl of TE buffer and stored at -200C freezer for
further analysis.
DNA re-precipitated with 100 µl of 3M sodium acetate and 2
volume of cold absolute ethanol.
Samples were treated with 1µl of Rnase-H and proteinase-K (1µl
each).
Fig No. 6 Procedure for DNA purification

19. Fig No. 7: Procedure for Nano Drop
(Gaikwad, 2015)
 Dilution of DNA-
A part of DNA sample was diluted with appropriate quantity
of sterilized distilled water to yield a working concentration of 15-
25 ng/l and stored at -200 C until PCR amplification.
19
DNA was quantified
1 µl DNA was loaded
TE (blank) was loaded
Blank was set
• By Nano Drop:

20.  ISSR analysis-
ISSR reactions were initially performed using 15 UBC primers
having 16-18 nucleotides long.
Table No. 4: Preparation of reaction mixture
20
Sr.
No.
Components
Final
concentration
s
Volume for one
tube
1. 2 X Master mix 2X 12.5µl
2. Primers 100 ng 1.0µl
3. Template DNA 15-25 ng 1.0µl
4. Nuclease free water - 10.5µl
Total 25µl

21. • The amplification was carried out in thermal cycler for 40 cycles
under following PCR conditions.
Table No. 5: Thermal cycling
• After the completion of PCR, the products were stored at -800C until
the gel electrophoresis.
(Zamanianfard et al., 2015)
21
Sr. No. Temperature Steps Time
1. 940 C Pre Denaturation 5 min
2. 940 C Denaturation
1 min
each
3. 46-500 C Annealing
4. 720 C Extension
5. 720 C Final Extension 10 min
40 cycles

22.  Resolution of amplified product on agarose gel
electrophoresis-
5 µl of PCR products and 2 µl bromophenol blue were loaded
on 2 % agarose. Gel stained with ethidium bromide to test the
success of PCR reactions. The sizes of amplified fragments were
determined using standard 1 kb and 100bp DNA ladder (Thermo
Scientific). Gel was photographed using a gel documentation
system.
(Deif et al., 2013)
22

23.  Stastical Analysis of DNA Fragments Pattern-
ISSR amplified bands were scored as present (1) absent (0) for each
primer population combination. Jaccard’s co-efficient of similarity was
calculated and a dendrogram was generated based on similarity co-
efficient by using unweighted paired group method of arithimatic
mean (UPGMA).
 Similarity Coefficient-
Greater the value of coefficient, compared variety will be more
similar to selected variety. Value of coefficient also signifies about the
extent of similarity between the two varieties.
23
zn

t
xy
n
n
(J)similaritysJaccard'
nxy= Number of bands common in sample a and b.
nt = Total number of bands present in all samples.
nz = Number of bands not present in samples.
(Al-Doss et al., 2009)

24.  Analysis of polymorphic percent-
The polymorphism information content (PIC) for each marker
was determined separately using the given calculation as under:
(Qadir et al., 2015)
24
100
BandsTotal
BandscPolymorphi
(%)PercentscPolymorphi 
Fig No. 8: PIC value calculation

25. Results and discussion
 Genomic DNA Extraction-
Total genomic DNA was extracted by using CTAB method.
Fig No. 9: Agarose gel electrophoresis of 22 genotypes of extracted genomic DNA
25

26.  DNA Purification-
DNA purification was done by using Proteinase-K and
RNase-H.
Fig No. 10: Agarose gel electrophoresis of 22 genotypes of purified genomic DNA
26

27.  DNA Quantification-
The concentration of extracted DNA was checked using
nanophotometer and was found out to be in the range of 198 to 700
ng/µl.
27
Sr.
No.
Genotypes
Concentration
(ng/µl)
At A260
/A280
Sr.
No.
Genotypes
Concentration
(ng/µl)
At A260
/A280
1 Ajantha (MAU) 415 2.08 12 WSM-163A 201 1.8
2 NIAW-1415 301 1.6 13 WSM-1757 315 1.9
3 NI-5439 215 2.0 14 WSM-1472 303 1.9
4 PKV (Washim) 298 1.9 15 WSM-109 201 1.7
5 AKW-3722 653 1.8 16 WSM-163 453 2.0
6 AKW-107 208 1.8 17 WSM-105 345 1.8
7 MACS-1967 205 1.8 18 WSM-42 248 1.9
8 LOK-1 198 1.9 19 WSM-55D 206 1.8
9 N-59 201 1.9 20 WSM-135 194 1.7
10 HD-2189 215 1.8 21 WSM-154A 209 1.8
11 HD-2781 700 1.9 22 WSM-174 367 1.9
Table No. 6: DNA concentrations of 22 wheat genotypes

28.  ISSR Analysis-
The ISSR reaction where initially perform using 15 UBC primers
having 15-18 nucleotides long.
 Suitable ISSR primers:
Series of UBC primers were used to evaluate polymorphism.
Universal random primers like UBC-807, UBC-809, UBC-8011, UBC-12,
UBC-13, UBC-16, UBC-23, UBC-31, UBC-37, UBC-857, and UBC-880
were used. 15 primers were screened, from this 11 primers were give
best polymorphism but 4 primers were not suitable for wheat.
 Band statistics:
Total 1204 ISSR amplified fragments were generated. The percent
of polymorphic bands was highest in UBC-813 (100%) and lowest in
UBC-823 (11.40%). The percent of monomorphic bands was highest in
UBC-823 (88.59%) while lowest was recorded from UBC-813 (0%).
Highest PIC value was observed in UBC-831 (0.91) and lowest was
observed in UBC-812 (0.60). 28

29. Sr.
No. Primers
Monomorphic
Bands
Monomorphic
percent(%)
Polymorphic
Bands
Polymorphic
percent(%)
Total
Bands
PIC Value
1 UBC-807 44 41.90 61 58.09 105 0.88
2 UBC-809 22 21.15 82 78.84 104 0.82
3 UBC-811 44 31.20 97 68.79 141 0.87
4 UBC-812 22 51.16 21 48.83 43 0.60
5 UBC-813 0 0 102 100 102 0.84
6 UBC-816 22 24.17 69 75.82 91 0.83
7 UBC-823 132 88.59 17 11.40 149 0.86
8 UBC-831 88 47.05 99 52.94 187 0.91
9 UBC-837 44 53.65 38 46.34 82 0.80
10 UBC-857 88 73.33 32 26.66 120 0.85
11 UBC-880 22 27.05 58 72.5 80 0.81
Total 528 43.85 676 56.14 1204
29
Table No. 7: Characteristics of amplified fragments obtained using 11 primers for
ISSR analysis of wheat genotypes
 Analysis of polymorphic percent (%)-

30.  PCR amplified products-
30
Fig No. 11: Fragments obtained from UBC-807 over 22 wheat genotypes
Fig No. 12: Fragments obtained from UBC-813 over 22 wheat genotypes

31. 31
Fig No. 14: Fragments obtained from UBC-857 over 22 wheat genotypes
Fig No. 13: Fragments obtained from UBC-823 over 22 wheat genotypes

32.  Data Analysis-
Cluster Analysis: Amplified fragments were scored for presence (1)
or absence (0) of band in each genotypes in Microsoft excel sheet
and this scored data were used in NTSYS-pc software v.2.2 for
cluster analysis.
32Fig No. 15: Dendrogram
A
B
A1
A2
B2
B1
B1a
B1b
A1a
A1b
A1b2
A1b1

33. Ajantha
NIAW-
1415
NI-
5439
PKV
AKW-
3722
AKW-
107
MACS-
1967
LOK-1 N-59
HD-
2189
HD-
2781
WSM-
163A
WSM-
1757
WSM-
1472
WSM-
109
WSM-
163
WSM-
105
WSM-
42
WSM-
55D
WSM-
135
WSM-
154A
WSM-
174
Ajantha 1.0
NIAW-
1415 0.70 1.0
NI-5439 0.75 0.61 1.0
PKV 0.78 0.82 0.74 1.0
AKW-
3722 0.76 0.62 0.81 0.66 1.0
AKW-
107 0.76 0.60 0.87 0.75 0.80 1.0
MACS-
1967 0.73 0.54 0.86 0.67 0.78 0.85 1.0
LOK-1 0.67 0.60 0.85 0.68 0.82 0.82 0.83 1.0
N-59 0.68 0.58 0.84 0.70 0.85 0.83 0.82 0.90 1.0
HD-2189 0.73 0.65 0.75 0.72 0.76 0.76 0.75 0.70 0.80 1.0
HD-2781 0.63 0.75 0.72 0.73 0.68 0.78 0.70 0.71 0.72 0.72 1.0
WSM-
163A 0.63 0.88 0.56 0.80 0.55 0.57 0.52 0.57 0.58 0.67 0.75 1.0
WSM-
1757 0.68 0.67 0.75 0.72 0.81 0.70 0.71 0.70 0.80 0.82 0.72 0.65 1.0
WSM-
1472 0.57 0.81 0.57 0.74 0.56 0.58 0.53 0.58 0.57 0.66 0.74 0.85 0.66 1.0
WSM-
109 0.62 0.74 0.71 0.78 0.70 0.72 0.64 0.67 0.66 0.73 0.74 0.74 0.75 0.84 1.0
WSM-
163A 0.72 0.57 0.76 0.64 0.77 0.75 0.81 0.64 0.70 0.74 0.64 0.55 0.81 0.63 0.72 1.0
WSM-
105 0.56 0.73 0.72 0.73 0.71 0.71 0.65 0.80 0.78 0.67 0.80 0.75 0.72 0.72 0.72 0.60 1.0
WSM-42 0.60 0.81 0.66 0.78 0.65 0.65 0.62 0.67 0.66 0.71 0.78 0.87 0.75 0.86 0.84 0.67 0.83 1.0
WSM-
55D 0.70 0.64 0.81 0.71 0.80 0.80 0.76 0.86 0.83 0.70 0.78 0.64 0.74 0.63 0.67 0.68 0.82 0.74 1.0
WSM-
135 0.73 0.56 0.80 0.61 0.78 0.74 0.84 0.74 0.75 0.80 0.70 0.56 0.75 0.60 0.66 0.78 0.67 0.66 0.81 1.0
WSM-
154A 0.80 0.61 0.77 0.70 0.74 0.76 0.82 0.76 0.75 0.84 0.67 0.61 0.71 0.62 0.64 0.72 0.63 0.62 0.72 0.84 1.0
WSM-
174 0.67 0.57 0.67 0.55 0.75 0.68 0.72 0.73 0.72 0.70 0.60 0.57 0.70 0.61 0.61 0.73 0.68 0.65 0.75 0.76 0.78 1.0
Table no. 08: Similarity matrix
32

34. The dendrogram divided into major 2 clusters A and B. Cluster A again
divided into A1 and A2. A1 again divided into their sub clusters A1a and
A1b. A1a contain Ajantha solitary genotype. A1b divided again into A1b1
and A1b2. A1b1 contains 10 genotypes viz., NI-5439, AKW-107, MACS-1967,
AKW-3722, LOK-1, N-59, WSM-55D, HD-2189, WSM-135 and WSM-154A.
A1b2 contains WSM-1757 and WSM-163 genotypes. A2 contain WSM-174.
Cluster B divided into B1 and B2. B1 again divided into B1a and B1b.
B1a contains 3 genotypes viz., NIAW-1415, WSM-163A, and PKV. B1b
contains 3 genotypes viz., WSM-1472, WSM-42, and WSM-109. B2 contains
HD-2781 and WSM-105.
Ajantha and WSM-174 are solitary genotypes. The maximum similarity
percentage i.e. 90% was found between Lok-1 and N-59. The minimum
similarity i.e. 52% was found between MACS-1967 and WSM-163A.
34

35. Summary and conclusion
The genomic DNA was isolated from 22 genotypes of wheat.
Extracted DNA was purified and quantified. PCR amplification of 22
genotypes was done using 15 ISSR primers. 11 primers were gave
polymorphism and 4 primers were not suitable for wheat genotypes.
The percent of monomorphic bands was highest in UBC-823 (88.59%)
while lowest was recorded from UBC-813 (0%). The percent of
polymorphic bands was highest in UBC-813 (100%) and lowest in UBC-
823 (11.40%). Highest PIC value was observed in UBC-831 (0.91) and
UBC-807 (0.88) and lowest was observed in UBC-812 (0.60).
To conclude that, this study determines the maximum
similarity i.e. 90% was found between Lok-1 and N-59. The minimum
similarity i.e. 52% was found between MACS-1967 and WSM-163A.
35

36. Future Prospectus
• Future thrust will be directed towards the holistic use of ISSR
primes for DNA fingerprinting, genetic analysis and linkage
mapping in wheat.
36

37. REFERENCEs
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(2009). Assessment of Genetic Diversity in Saudi Wheat
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• Chaudhary, N., Kajla, S., Poonia, A., Panwar, S., and Khatkar, B.
(2016). Assessment of Molecular Diversity Using ISSR Markers for
Characterization of Wheat Varieties. International Journal of
Innovative Research in Science, Engineering and Technology.
5(6):10395-10941.
• Deif, M., Rashed, M., Sallam, M., Mostafa, E., and Ramadan, W.
(2013). Characterization of Twenty Wheat Varieties by ISSR
Markers. Middle-East Journal of Scientific Research. 15(2):168-175.
37

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39

40. • Qadir, A., Ilyas, M., Akhtar, W., Aziz, E., Rasheed, A., and
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40

41. Thank You
41