III Materials and methods

The investigation on “Assessment of Induced Genetic Variability in

Jatropha curcas L. through Molecular markers” was carried out at Department of

Agricultural Biotechnology, Anand Agricultural University, Anand.

3.1 Experimental Material:

Seeds of Jatropha curcas L. were exposed to various doses of gamma rays i.e. 0,

1, 3, 5, 10, 15, 20, 25 and 50 KR. Population of plants grown from treated seeds was

studied and mutants were selected on basis of morphological (height) variations from

experimental plantations raised at the Jatropha farm of Anand Agricultural University,

Anand.(Table 3.1). The plants grown from seeds without treatment of gamma rays were

taken as “Control”. Four mutants for each dose were taken for the investigation.

3.2 Glass-war

Sr. No. Dose (Kr) Sr. No. Dose (Kr)

Control 0

1 1 17 15

2 1 18 15

3 1 19 15

4 1 20 15

5 3 21 20

6 3 22 20

7 3 23 20

8 3 24 20

9 5 25 25

10 5 26 25

11 5 27 25

12 5 28 25

13 10 29 50

14 10 30 50

15 10 31 50

16 10 32 50

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Table 3.1: List of Mutants:

3.2 Glass-ware, Plastic-ware and Reagents:

The glass-wares and plastic-wares used were from Schott Duran, Germany and

Axygen, respectively and all the chemicals and reagents used in the present study were of

biotechnology or molecular biology grade quality.

3.3 DNA Extraction :

Total genomic DNA was extracted from the leaves of standing crop by Cetyl

trimethyl ammonium bromide (CTAB) method (Dakshinamoorthy and selvaraj,2000)

with minor modifications.

3.3.1 Preparation of stock solutions for reagents and buffers for DNA extraction

The reagents and buffers for DNA isolation were prepared as per Sambrook

et al., (1989). The composition and procedure for preparation of various stock solutions

and buffers are given in Table 3.2.

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Table 3.2: Preparation of stock solutions for DNA extraction and electrophoresis

Sr. No Solution Method of preparation

11M Tris HCl (pH 8.0)

100 ml

12.11 g Tris base (Himedia) was dissolved in 80 ml

distilled water. The pH was adjusted to 8.0 by adding

concentrated HCl and the total volume was adjusted to

100 ml. It was dispensed into reagent bottle and

sterilized by autoclaving.

20.5M EDTA (pH 8.0)

100 ml

18.60 g EDTA di Sodium salt (Himedia) was

Dissolved in 80 ml distilled water. The pH was

adjusted to 8.0 by adding NaOH pellets. The total

volume was adjusted to 100 ml and dispensed into a

reagent bottle and sterilized by autoclaving.

3 5M NaCl, 100 ml

29.22 g NaCl (Himedia) was taken in to beaker; 50 ml

of distilled water was added and mixed well. When the

salts got completely dissolved, the final volume was

adjusted to 100 ml. It was dispensed into a reagent

bottle and sterilized by autoclaving.

4 80% Ethanol, 100 ml

80 ml of ethanol was taken and 20 ml of distilled

water was added, mixed well and dispensed into a

reagent bottle and stored at 40C.

5Chloroform: Isoamyl

alcohol (24:1), 100 ml

96 ml of chloroform (Qualigens) and 4 ml of isoamyl

alcohol (Qualigens) were measured, mixed well and

stored in a reagent bottle at room temperature.

6Ethidium Bromide

(10 mg/ml), 1.0 ml

10 mg Ethidium Bromide (Himedia) was added to 1.0

ml of distilled water and it was kept on magnetic

stirrer to ensure that the dye has dissolved completely.

It was dispensed into an amber colored eppendorf tube

and stored at 40C.

7 1X TE buffer, 100 ml

10mM Tris HCl

(pH 8.0)

0.1mM EDTA

1.0 ml of Tris HCl (1M), 200 µl of EDTA (0.5M)

were taken and distilled water was added to adjust the

final volume of 100 ml, mixed thoroughly, autoclaved

and stored at room temperature.

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(pH 8.0)

8TBE buffer 5X

(1 liter) pH 8.0

54.5 g of Tris base, 27.5 g of Boric acid (Qualigens)

were taken, 20 ml of 0.5M EDTA (pH 8.0) was added.

The Final volume of 1 liter was adjusted by adding

distilled water and the pH was adjusted to 8.0.

93M Sodium acetate,

20 ml pH 7.0

8.16 g Sodium acetate trihydrate salt (Merck) was

dissolved in distilled water and the final volume was

made up to 20 ml. The pH was adjusted to 7.0.

Table 3.3: Preparation of Extraction Buffer

Buffer Method of preparation

CTAB extraction buffer

(5%), 10 ml

1.0 ml of 1M Tris HCl (pH 8.0), 2.8 ml of 5M NaCl, 1 ml

of 0.5 M EDTA (pH 8.0) ,0.5 gm of CTAB (w/w) , 0.2 gm

of PVP (Polyvinyl pyrrolidine)(w/w) and 5.2 ml of

distilled water were taken in to a flask and mixed well.The

buffer was kept incubated 1t 650C for the CTAB and PVP

to get dissolved. 0.15 ml (1.5%) β- mercaptoethanol

(Qualigens) was added into the mixture just before use.

3.3.2 Protocol for Genomic DNA extraction:

Total DNA was extracted from the leaves by Cetyl trimethyl ammonium bromide

(CTAB) method (Dakshinamoorthy and Selvaraj, 2000) with some modifications as

follows:

The leaves of Jatropha germplasm were directly collected from field and

utilized for DNA extraction.

Weighing leaf sample from each accession, 300 mg was grinded in liquid

nitrogen using mortar and pestle.

CTAB buffer 5% (1 ml) containing 1.5% (v/v) β-mercaptoethanol (added

fresh in extraction buffer) and 15 μl Proteinase k (Fermentas, USA) was

quickly added to each microcentrifuge tube (2 ml) and vortex to mix.

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The tube was incubated at 65°C for 1-1/2 hr with frequent swirling. An

equal volume of chloroform : isoamylalcohol (24:1) (chilled) was added and

centrifuged at 10,000 rpm (Eppendorph 5417R) and 4°C for 10 min to

separate the phases.

The supernatant was carefully decanted and transferred to a new tube.

The above steps, beginning with the addition of chloroform: isoamylalcohol

(24:1) and ending with decanting of supernatant, were repeated twice.

The supernatant was precipitated with double volume of absolute ethanol

or 0.6 volume of iso-propanol along with 100 µl of 0.3 M sodium acetate for

overnight at 40C. The precipitated nucleic acids were collected and washed

once with the 70% ethanol and once with 80% ethanol. (The tubes should not

be shaken vigorously, because DNA is very vulnerable to fragmentation at this

step).

The pellets were air dried and resuspended in 100 µl of TE buffer (10 mM

Tris-HCl; pH 8.0, 0.1mM EDTA, pH 8.0).

DNase free RNase A (Fermentas, USA) 5μl was added to the dissolved

DNA stock and incubated in a water bath at 370C for 1 hour followed by 600C

for 10 minutes for enzyme inactivation. The samples were stored at -200C

deep freeze for long-term usage.

3.3.3. Purity and Quantification test of DNA

Spectrophotometry was performed to determine DNA concentration by

using Nanodrop N.D.1000 (Software V.3.3.0, Thermo Scientific, USA) at absorbance

ratio 260/280 nm and the quality of obtained DNA was checked on 0.8% agarose gel.

Dilution of 20 ng/µl working solution was prepared from the stock solution of the isolated

DNAs.

3.3.3.1 Procedure:

The 1.5 μl of DNA sample was loaded into the well of Nanodrop

Spectrophotometer (Thermo Scientific, U.S.A.) and the concentration of DNA and

absorbance at 260 nm and 280 nm were measured and the A260/A280 ratio was

automatically calculated by the software. 19 | P a g e

Table 3.4: Preparation of working solution of DNA(20ng/µl, 100µl) for RAPD and

DAMD analysis :

Sr.

No

Mutants Stock solution taken

(µl)

Water(Nuclease free) added

(µl)

C Control 1.4 98.6

1 1Kr1 8.5 91.5

2 1Kr2 1.3 98.7

3 1Kr3 2.1 97.9

4 1Kr4 2.1 97.9

5 3Kr1 2.2 97.8

6 3Kr2 1.5 98.5

7 3Kr3 2.8 97.2

8 3Kr4 9.3 90.7

9 5Kr1 5.1 94.9

10 5Kr2 4.5 95.5

11 5Kr3 3.6 96.4

12 5Kr4 5.4 94.6

13 10Kr1 4.2 95.8

14 10Kr2 4.8 95.2

15 10Kr3 6.9 93.1

16 10Kr4 4.2 95.8

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17 15Kr1 5.7 94.3

18 15Kr2 7.2 92.8

19 15Kr3 2.8 97.2

20 15Kr4 4.8 95.2

21 20Kr1 5.0 95.0

22 20Kr2 5.5 94.5

23 20Kr3 5.3 94.7

24 20Kr4 2.5 97.5

25 25Kr1 1.7 98.3

26 25Kr2 1.0 99.0

27 25Kr3 1.7 98.3

28 25Kr4 0.9 99.1

29 50Kr1 4.8 95.2

30 50Kr2 4.1 95.9

31 50Kr3 2.7 97.3

32 50Kr4 5.0 95.0

3.4 Randomly Amplified Polymorphic DNA (RAPD):

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Amplification of RAPD fragments was performed according to

Williams et al., (1990) with some modifications using decamer arbitrary primers (MWG

Biotech, Germany) (Table 3.6).

3.4.1. RAPD PCR Components

The reagents and list of Primers (MWG biotech,Germany) used for RAPD-PCR

amplification of DNA were as follows(Table 3.5,3.6)

Table 3.5: Components of PCR reaction mixture

Table 3.6: List of RAPD primers used for the present study

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Sr.No Reagents Volume

1 PCR Taq Buffer with MgCl2 (Bangalore Genei,India) 2.5 µl

2 Primer (10p moles/ µl)(MWG biotech,Germany) 1.5 µl

3 dNTPs (2.5mM each)(Fermentas,USA) 0.5 µl

4 Taq Polymerase (3U/ µl) (Bangalore Genei,India) 0.5 µl

5 Template DNA (20ng/ µl) 2.5 µl

5 Nuclease free water (Amresco,USA) 17.5 µl

Total 25 µl

Sr. No. Primer name Sequence (5’ 3’) GC content (%)

1OPA09 GGGTAACGCC 70

2 OPA-18 AGGTGACCGT 60

3 OPB10 GTGACATGCC 60

4 OPB11 AAGACCCCTC 60

5 OPC18 CAGCTCACGA 60

6 OPD05 ACCAGGTTGG 60

7 OPF04 GGATGAGACC 60

8 OPF10 TGGACCGGTG 70

9 OPH12 ACGCGCATGT 60

10 OPH13 GACGCCACAC 70

11 OPI09 TGGAGAGCAG 60

12 OPI10 ACAA GCGAG 60

13 OPI13 CTGGGGCTGA 70

14 OPI15 TCATCCGAGG 60

15 OPK16 GAGCGTCGAA 60

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3.4.2 Cocktail preparation:

As per the above cocktail, Millipore sterilized water was added first followed by

addition of above mentioned PCR reagents in sequence and finally the template DNA.

The reaction mixture was prepared in 200 µl PCR tubes (Axygen, USA). The reagents

were mixed gently by tapping against the tube followed by a short spinning (~3,000 rpm

for 60 seconds). The tubes were then placed in the thermal cycler (Biometra,T-Gradient

Germany) for cyclic amplification with the following PCR reaction conditions ( Figure

3.1) PCR conditions (Williams et al, 1990).

940 940 720 720

4 min 1 min 2 min 7 min

380

1 min 40

α

Figure 3.1 : Steps in PCR amplification reaction conditions

3.4.3 Electrophoresis of the amplified product:

The amplified products of RAPD were analysed on 1.5% agarose gel.

3.4.3.1 Chemicals used:

Agarose (Low EEO,Bangalore Genei,India)

5X Tris Borate EDTA (TBE) Buffer ,pH 8.0

Ethidium Bromide (EtBr) (10 mg/ml)(Amresco,USA)

Gel Loading Dye (6X) (Fermentas,USA)

100 bp DNA ladder (fermentas,USA)

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Annealing

H

old

42 cycles

Hold

Denaturation

Initial

denaturation ExtensionFinal

extension

Agarose gel of 1.5 % concentrations was prepared in 1X TBE (1.5 g agarose in

100 ml 1X TBE and 2.5 µl Ethidium bromide from 10 mg/ml stock). PCR amplified

products (9 µl and 1 µl 6X loading dye) were loaded into the wells. The 100 bp standard

DNA ladder (1 µl) (marker) was also run along with the samples. The electrophoresis was

conducted at 120 V current (constant) for 3 hrs. to separate the amplified bands. The

separated bands were visualized under UV transilluminator (Biometra,Germany) and

photographed using gel documentation system (Bio-rad,California).

3.5: Direct Amplification Of Minisatellite DNA (DAMD markers)

The amplification of Genomic DNA was carried out using four universal

DAMD primers which were utilized by Ranade et al., (2007) in their studies related to

diversity assessment in Jatropha curcas.The amplification was carried out according to

the method followed by Heath et al.,(1993) with some modifications. The reaction

components ( table 3.7) and the list of primers used in the present study (table 3.10) are

as follows.

Table 3.7: PCR reaction components

Sr.No Reagents Volume

1 PCR Taq Buffer with MgCl2 (Bangalore Genei,India) 2.5 µl

2 Primer (10p moles/ µl)(MWG biotech,Germany) 1.5 µl

3 dNTPs (2.5mM each)(Fermentas,USA) 0.5 µl

4 Taq Polymerase (3U/ µl) (Bangalore Genei,India) 0.5 µl

5 Template DNA (20ng/ µl) 2.5 µl

5 Nuclease free water (Amresco,USA) 17.5 µl

Total 25 µl

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Table 3.8: List of DAMD primers (Ranade et al.,,2007)

Primer Name Primer Sequence GC content (%) Tm value(0C)

HBV GGTGTAGAGAGGGGT, 60 50.6

HVR CCTCCTCCCTCCT 69.2 44.0

33.6 GGAGGTTTTCA, 45.5 32.0

M13 GAGGGTGGCGGTTCT, 66.7 53.3

All the PCR reactions were carried out in 0.2 ml capacity thin walled PCR tubes.

( Tarson,, India). As per the above cocktail, Millipore sterilized water was added first

followed by addition of PCR reaction components, primer in sequence and finally the

template DNA. The reagents were mixed gently by tapping against the tube followed by a

short spinning (~3,000 rpm for 60 seconds). The tubes were then placed in the Thermal

Cycler (Biometra T-Gradient, Germany) for cyclic amplification (Figure 3.2).

940 940 720 720

4 min 1 min 2 min 7 min

490,420,360,500

1.05 min 40

α

Figure 3.2: PCR reaction conditions for DAMD amplification

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Annealing

Hold

Initial denaturation

Denaturation Extension Final extension

3.4.1: Electrophoresis of PCR amplified products

The amplified products of ISSR were analyzed using 1.8 % agarose gel.

3.4.1.2: Chemicals used

Agarose (Low EEO,Bangalore Genei,India)

5X Tris Borate EDTA (TBE) Buffer ,pH 8.0

Ethidium Bromide (EtBr) (10 mg/ml)(Amresco,USA)

Gel Loading Dye (6X) (Fermentas,USA)

100 bp DNA ladder (Fermentas,USA)

Agarose gel of 2.0 % concentration was prepared in 1X TBE (2.0 g agarose in 100

ml 1X TBE and 2.5 µl Ethidium bromide from 10 mg/ml stock). PCR amplified products

(12 µl and 1.5 µl 6X loading dye) were loaded into the wells. The 100 bp standard DNA

ladder (1 µl) (marker) was also run along with the samples. The electrophoresis was

conducted at 120 V current (constant) for 3 hrs. to separate the amplified bands. The

separated bands were visualized under UV transilluminator (Biometra,Germany) and

photographed using gel documentation system (Bio-rad,California).

3.5: Data analysis

Clear and distinct bands amplified by RAPD and DAMD and primers were scored

for the presence and absence of the corresponding band among the genotypes. The

scores 1 and 0 indicates the presence or absence of bands respectively. The softwares

used for the analysis of the scored data were NTSTSpc version 2.02 (Rholf 1998),

Popgene 32 (Yeah et al.,1999) and SPSS .The major part of the analysis was carried out

using NTSYSpc version 2.02 (Rholf,1998) except for the calculation of the Shannon

index, observed and effective number of alleles which were calculated using Popgene

32.

3.5.1 Genetic similarity and cluster analysis

Coefficients of similarity were calculated by using Jaccard’s similarity coefficient

by SIMQUAL function and cluster analysis was performed by agglomerative technique

using the UPGMA (Un-weighted Pair Group Method with Arithmetic Mean) method by

SAHN clustering function of NTSYS-pc. Relationships among the Jatropha curcas

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genotypes and among and between the species of jatropha genus were expressed in the

form of dendrograms and genetic similarity matrix. .

3.5.2 Cophenetic correlation and mantel tests

The cophenetic correlation analysis was carried out using the COPH function of

NTSYS-pc. In this method dendrogram and similarity matrix were correlated to find the

goodness-of-fit of the dendrogram constructed based on similarity coefficients. The

correspondence between RAPD and DAMD based on similarity coefficient matrices was

tested using cophenetic correlation analysis and Mantel matrix correspondence test. The

Mantel matrix correspondence test was carried out using the MXCOMP function in the

NTSYSpc version 2.02.

3.5.3 Principal Component Analysis

Principal component analysis was carried out using the EIGEN module of

NTSYSpc 2.02.The results were graphically expressed in the form of 2D and 3D plots

generated by graphics module after the calculation of EIGEN values. First three eigen

values which showed maximum variation were extracted as principal components and

2D and 3D plots were generated on that basis.

3.5.4 :Calculation of parameters of genetic variability

Various components were calculated which included, No of monomorphic and

polymorphic loci , Polymorphism Information Content (PIC),Effective Multiplex ratio

(EMR), Polymorphism %. Polymorphism Infromation content (PIC) was calculated

according to formula

described by Smith et al (1997) ,Bootstein et al.,(1980) Garcia et al (2004).

PIC=1-∑f2 where f is the frequency of ith allele.

All the above mentioned variables were calculated individually for both the

markers as well as the combined values for RAPD+DAMD were calculated for

comparing ability of the markers for DNA polymorphism assessment.

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