MATERIALS AND

METHODS

MATERIALS AND METHODS

The present investigation entitled Molecular Characterization of Desi Cotton
Hybrids and Their Parents was carried out at the Main Cotton Research Station, Navsari
Agricultural University (NAU), Athwa Farm, Surat. This chapter contains thedetails
regarding the experimental materials and methodology adopted during the courseof
investigation.
3.1Experimental Material
The experimental material of cotton genotypes were procured from the Main Cotton
Research Station, Navsari Agricultural University, Athwa Farm, Surat. The details of
genotypes included in the present investigation are summarized in Table 3.1.1
Table No. 3. 1.1: List of hybrids and their parents used in the present study.
HBRIDS

FEMALE PARENT

MALE PARENT

G.cot.DH-7

Sujay

G.27

G.cot.DH-9

4011

824

3.1.2 Laboratory ware

All glass ware used were obtained from Borosil and disposable plastic
ware(centrifuge, tubes, PCR tubes, micro tips) from Eppendorf.
3.1.3 Chemicals
All the chemicals used were of analytical reagent grade/molecular grade and were
obtained from Hi-Media/ E-Merck/Qualiges and Bangalore Genei.

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3.1.4 Equipments:
NAME OF INSTRUMENTS
Weighing balance
Laminar flow
Camera
pH meter
Spectrophotometer
Water bath
Electrophoresis unit
Micropipettes
Centrifuge
Hot Air Oven
Gel documentation
Thermo Cycler
Dry bath

COMPANYNAME
SHIMADZU
MAC, Delhi
Sony
GeNei
EI
GeNei
GeNei
Eppendrof
Dynamica
EIE
UVTEC (Cambrige)
BIORAD
GeNei

3.2 Extraction of plant genomic DNA

Genomic DNA isolated from young fully expanded leaves ofhybrids and their
respective parents using the CTAB method with minor modification. (Sanghai Maroof et al.,
1984).
Materials:

CTAB extraction buffer

Liquid nitrogen

Chloroform: Isoamylalcohol (24:1)

Isopropanol (prichilled at-20C)

70% Ethanol

T10E1

3.2.1Procedure:

100 mg of leaf tissue was taken from the young plant. The leaves were surface
sterilized and washed with distilled water. The sample was ground to fine powder
using liquid nitrogen with pre chilled pestle and mortar.

The powder was transferred into a sterile 2.0 ml eppendorff tube. Then add 1 ml
prewarmed CTAB extraction buffer [1M Tris (pH 8.0), 0.5 M EDTA (pH 8.0), 5 M
NaCl, 0.01 M -mercaptoethanol, 3% CTAB, 1% PVP].

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The tubes were vortexed and incubated at 65C for 1 hr with intermediate mixing.

Equal volume of Chloroform: Isoamylalcohol (24:1) was added, mixed by mild

vortexing for 5 to 10 sec and centrifuge at 13000 rpm for 15 min at room temperature.

Supernatant (upper phase) was collected and add equal volume of Chloroform:
Isoamylalcohol (24:1) in new tubes, mixed well and centrifuged at 15000 rpm for 15
min at 4C.

The top aqueous phase was transferred to another 1.5 ml tube and adds equal volume
of ice cold isopropanol, mixed well and kept at -20C for 30-45 min.

After this DNA was pelleted by centrifugation at 15000 rpm for 15 min at 4C.

Discard supernatant add 500l of 70% ethanol. Again centrifuge at 14500 rpm for 3
min at room temperature.

Pellet was recovered, air dried and redissolved in 100 ml T10E1 buffer.

Then add 2l of RNase (10 mg/ml) to the DNA tube and incubated at 37C in water
bath for 30 min.

Add equal volume of Chloroform: Isoamylalcohol (24:1) and centrifuged at 15000

rpm for 15 min at 4C.

The top aqueous phase was transferred to another 1.5 ml tube and equal volume of ice
cold isopropanol, mixed well and kept at -20C for 30-45minutes.

After this DNA was pelleted by centrifugation at 15000 rpm for 15 min at4C.

The pellet was washed with 70% ethanol and centrifuge at 14500 rpm for 3 min at
room temperature.

Discard supernatant and pellet was recovered, air dried and redissolved in 100 ml
T10E1 buffer.

The DNA was stored at -20C for further use.

3.3 Quantification of genomic DNA

To estimate the quality and quantity (in terms of protein and RNA contamination) of
isolated genomic DNA, spectrophotometry was performed. DNA (2l) was loaded in the well
of Nanodrop instrument and the concentration of DNA and absorbance was taken at 260 to

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280 nm were measured as well as the A260 /A280 ratio was calculated. The conversion factor or
constant used was 50 for concentration measurement.
3.4 Quality assessment of DNA by agarose gel electrophoresis
Materials:

Agarose powder

1X TBE buffer

Ethidium bromide

Electrophoretic unit

6X gel loading dye

3.4.1 Procedure:
The quality of DNA was assessment on 0.8% (W/V) agarose gel prepared in 100 ml
1X TBE contains 3l Ethidium bromide (Etbr). The already extracted genomic DNA from
stock (8l) was mixed with 2l of 6X gel loading dye. 7l sample was loaded in each well
using micropipette. The gel was provided with potential difference of 100 V. The bands were
visualized under UV light using transilluminator. The sample which resolved into single
discrete high molecular weight band near the well with no shearing, were considered to be of
good quality.

3.5 Dilution of DNA

For PCR reaction, the extract DNA was diluted to 10 ng/l for RAPD and ISSR
analysis and 60 ng/l concentrations for SSR analysis using TE buffer.
3.6 Molecular analysis of DNA
3.6.1 RAPD analysis
The genomic DNA is subjected to PCR. A total of Twenty five oligonucliotide
primers GeNei) were used for RAPD analysis. RAPD with 25l reaction volume were carried
out in 200l PCR tube. Each reaction mixture (25l) for PCR amplification consisted of 10X
assay buffer (10mM Tris-HCl, pH 8.3 and 50mM KCl), 25mM MgCl2, 3U Taq DNA
polymerase; 2.5 mM each dATP, dCTP, dGTP, dTTp (Banglore GeNei), 100ng/l decamer
primer(Banglore GeNei), and 10ng genomic DNA template.

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Table 3.2: Composition of master mix of RAPD

Components

Quantity

Sterile distilled water

17l

10X assay buffer

2.5l

2.5mM MgCl2

1l

25mM dNTPs

1l

100ng/l primer

1l

3U/l Taq DNA polymerase 0.5l

10ng/l Template DNA
Total

2l
25 l

Table 3.3: List of RAPD primers

Sr.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13

Nameof primers

Accession numbers

RPI 1
RPI 2
RPI 3
RPI 4
RPI 5
RPI 6
RPI 7
RPI 8
RPI 9
RPI 10
RPI 11
RPI 12
RPI 13

AM765819
AM750044
AM773310
AM773769
AM773770
AM773771
AM773312
AM773773
AM773315
AM750045
AM911709
AM773316
AM750046

Sr.
No.
14
15
16
17
18
19
20
21
22
23
24
25

Nameof
primers
RPI 14
RPI 15
RPI 16
RPI 17
RPI 18
RPI 19
RPI 20
RPI 21
RPI 22
RPI 23
RPI 24
RPI 25

Accession
numbers
AM773774
AM773775
AM773776
AM911710
AM765830
AM773777
AM773317
AM765820
AM911711
AM911712
AM765821
AM750054

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3.6.1.1 PCR Programming for RAPD

An initial denaturing step of 5 min at 94C, 30 cycles consisting each of a denaturing
step of 45 sec at 94C, primer annealing step of 1 min at 35C and primer extension step of
1min at 72C were given. Final step of 10 min at 72C was given for polishing the end of
PCR products. PCR amplification was carried out in thermal cycler(BIORAD).
3.6.1.2Electrophoresis of amplified DNA
Amplified DNA fragments were separated on 1.5 % agarose gels and stained with
ethidium bromide. Running buffer containing TBE [Tris-buffer, boric acid and EDTA(pH
8.0)] was used for electrophoresis. Wells were loaded with 20 l of a sample and 5l of
loading buffer (sucrose, bromophenol blue and Xylene cyanol). The gel was run at80 V
(constant) to separate the amplified bands. The standard DNA marker (100 bp) was also run
along with the samples. The separated bands were documented under UV transilluminator
and photographed by Gel documentation system (AlphaInnotech.U.S.A.) and analyzed.

3.6.2 ISSR analysis

Nineteen oligonucleotide ISSR primers were used for hybrid analysis. ISSR with 25l
reaction volume were carried out in 200l PCR tube. Each reaction mixture (25l)for PCR
amplification consisted of 1X reaction buffer (10 mM Tris-HCl, pH 8.3 and 50mMKCl), 2.5
mM MgCl2, 3U Taq DNA polymerase; 2.5 mM each dATP, dTTP, dCTP and dGTP (all
reagents from Fermentas), 10 pico mole primer (Bangalore GeNei India),and 10 ng genomic
DNA template.
Table 3.4: Master mixture of ISSR
Components
Sterile distilled water
10X assay buffer
2.5mM MgCl2
10mM dNTPs
3U/l Taq polymerase
10 p mol primer
60 ng/l template
Total

Quantity(l)
17
2.5
1
1
0.5
1
2
25

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Table 3.5: List of ISSR primers sequence

Sr No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

Oligo name
ISSR 1
ISSR 2
ISSR 3
ISSR 4
ISSR 5
ISSR 6
ISSR 7
ISSR 8
ISSR 9
ISSR 10
ISSR 11
ISSR 12
ISSR 13
ISSR 14
ISSR 15
ISSR 16
ISSR 17
ISSR 18
ISSR 19

Sequence (5-3)
AGCAGCAGCAGCAGCGA
AGCAGCAGCAGCAGCGG
AGCAGCAGCAGCAGCGT
AGCAGCAGCAGCAGCGC
CACACACACACACAAT
CACACACACACACAAC
CACACACACACACAGT
CACACACACACACAGC
CACACACACACACAGA
CACACACACACACAAA
GTGTGTGTGTGTGTTA
GTGTGTGTGTGTGTTG
GTGTGTGTGTGTGTCA
GTGTGTGTGTGTGTCT
GTGTGTGTGTGTGTAT
GTGTGTGTGTGTGTAC
CAGGAGAGAGAGAGAA
GCTGAGAGAGAGAGAGA
GCAGAGAGAGAGAGAGA

Tm(C)
48
48
48
58
46
48
48
50
48
46
46
48
48
48
46
48
52
52
52

3.6.2.1 PCR programming for ISSR:

An initial denaturing step of 5 min at 94C, 45 cycles consisting each of a denaturing
step of 1 min at 94C, primer annealing step of 45 sec at 42C and primer extension step of 2
min at 72C was given. Final step of 5 min at 72C was given for polishing the ends of PCR
product. PCR amplification was carried out in Thermal cycler (biored). The amplified
product was separated by electrophoresis and polymorphism was detected on 1.5% agarose
gel by using ethidium bromide.
Table 3.6: PCR amplification steps for ISSR
Stage
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Hold

Tm (C)
94
94
55
72
72
4

Duration
5 min
1 min
45 sec
45 sec
10 min

Cycles
1
40
1

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3.6.3 SSR Analysis

A total 40 SSR primers, (MGHES, JESPR Series, Euro fins Genomics India Put Ltd)
were used for the study. PCR reaction(25l) mixture contained DNA, 60 ng/l; 10X reaction
buffer; 10mM dNTPs; 2.5mM MgCl2; 10 micromoles each of forward and reverse primers.
The amplification was carried out in thermal cycler.
Table 3.7: Master mixture of SSR
Components

Quantity (l)

Sterile distilled water

16

10X assay buffer

2.5

2.5mM MgCl2

25nm dNTPs

10 p mol Forward primer

10 p mol Reverse primer

3U/l Taq polymerase

0.5

Template DNA

Total

25

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Table 3.8: List of SSR primer sequencer

Sr
No.

Primer
name

Reverse primer

Forward primer

Tm
(C)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

JESPR-223
JESPR-228
JESPR-230
JESPR-231
JESPR-232
JESPR-234
JESPR-296
JESPR-298
JESPR-300
JESPR-307
JESPR-153
JESPR-291
TMB 409
TMB 1268
BNL 786
BNL 3090
CM 45
CM 56
NAU 2083
MGHES 33
MGHES 62
MGHES 66
JESPR -6
JESPR-286
JESPR-209
TMB 283
TMB 327
TMB 1181
TMB 1615
TMB 1791
CIR 61
CIR 216
BNL 1531
BNL 1053
BNL 1721
BNL 3649
BNL 1317
BNL 1414
BNL 686
BNL 3408

CGTTSCGGATTATTGGACATG
GGCAAGCAAAGCAAAACTC
GAAACCCTTGGCCATGAG
CTATGAACTGCTGGCTATGG
CGTTGTATTATTATTTCCAGTGCTCG
CTAACTCGAATCCGTCAC
TGACCTCAATTTAGAAACCC
GGACCTTCGGAATAATTACC
CGGAAAATGATGATGATGAGAAG
GAAAGACACTAAGCTGAGGC
GATTACCTTCATAGGCCA
CATTCCCCACTTTGCTCT
CAGAGGACGAAGGTAGC
CAGGTACCATTGATGCCA
CTTTCCACGTGTAATTTG
GAAATCATTGGAAGAACA
GATGCCAGTAAGTTCAG
CAATCATAGACCGCCGCCACA
AGAAGAGGTTGACGGTC
TTTTTGGGCTTTCTTTTCT
TGCATCTGATCTAATTGT
TCCTCCTCCCACTTCATC
ACTAAACCCTAAACACAA
GGAGGACATGGGTTTGA
ATTGAGAGGCATTTTGGT
GGCTCCAAAATTGAAACG
GCAATGTCTTCACCAG
CATCTTCAATATTCACCAG
TTGATTGATAACGCGACA
GAGATCGTTTATCATTCC
TTAGTCCTCTACATACCG
ATCTGAACCATCATCCTC
CTGCAACAAGAGCCTGT
AGGGTCTGTCATGGTTG
TGTCGGAATCTTAAGACC
GCAAAAACGAGTTGACC
AAAAATCAGCCAAATTGG
AAAAACCCTTTCCAAGA
ATTTTTCCCTTGGTGGTCCT
ATCCAAACCATTGCACCACT

TGGTCCAAAGCTCAAG
CAGAACAACACCATCAACACTCTCAG
GGGACTAAAGAAGTAATTATGCC
GCTGGTGGGATTCTCTG
CAGACCACGCTATTTTTGCC
GCATAGTTATGAATGACTCTC
GGGTGTTACATAGAGTGTATAAAATTG
GATGCCCTCGTGTTAAAG
CGCATCACAAACCAAACAC
CTTGGCCATGTATTCCTTCA
GAAAACATGAGCATCCTG
CATGTTTCTTTGCCCATC
TGGTGGGTTTCACTTTCA
CTCGAAACCTAGTGC
GATCTTAACTCTTGCTCT
TTGCTCCGTATTTTCCAG
GCCAACTTATATTCGGTT
ACATTGCTAGATATTATA
TGAGTGAAGAACCTGCA
CCAATTACGCATGTTCAA
TGTTCCTCACAGCAAGAG
GACTGTGGCTGGAGGAG
CATTATAAGGTCCCCAAT
GCATGCATGTAAAATGTA
AGATGACAAAAATTGTG
GTGGACATTGGCATTCAT
ATTGCCGACTTTGCCTGT
ATTGCCGACTTTGCCTCT
CAGCGTTGAACTATCCAC
TCTACCGGCCTCGTAAG
TCATAATAAAGGCGTGG
TTCTGATTGGCACTTTC
ATGGAGATTGGCTGAGAT
CATGCATGCGTACGTGT
GCGCAGATCCTCTTACCA
CCTGGTTTTCAAGCCTGT
CGTCAACAATTGTCCCAA
GGTGTCCTTCCCAAAAATT
ACATTGATAGAAATATAAACCAAACACG
GTGTACGTTGAGAAGTCCA

55
55
55
55
55
55
55
55
55
55
56
56
56
56
56
56
56
56
56
55
55
55
55
55
55
55
55
55
55
55
55
55
56
56
56
56
56
56
56
56

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3.6.3.1 PCR Programming for SSR

An initial denaturation step of 5 min at 94C followed by 30 cycles consisting each of
a denaturing step of 1 min at 94C, primer annealing step of 45 sec at 55C, and primer
extension step of 1 min at 72C were given. The program ended with one final extension at
72C for 10 min. The amplified products were resolved on 3% agarose gel with ethidium
bromide.
Table 3.9: PCR programming of SSR
Stage

Tm(C)

Duration

Cycles

Initial denaturation

94

5 min

Denaturation

94

1 min

Annealing

55

45 sec

Extension

72

45 sec

Final extension

72

10 min

Hold

30

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