3.1 Plant material and DNA extractionshodhganga.inflibnet.ac.in/bitstream/10603/6271/8/08_chapter...

TERI School-Ph.D.Thesis, 2004

Materials and Methods

3.1 Plant material and DNA extraction

3.1.1 Plant Material

The plant germplasm was mainly collected as leaf samples. The leaf material

collected for different objectives are as follows:

3.1.1.1 Genetic diversity analysis in P.deltoides

Leaf material of 43 P. deltoides clones introduced in India from various regions

of the world was collected. The source from where these clones were obtained

for introduction in India was identified for most of the clones except few whose

original source were not known. Leaf samples were collected from forty-three

P. deltoides genotypes and P. ciliata, P. maximowiczii and P. euphratica

genotypes which were included as controls in the study. P. deltoides and

P. ciliata leaf material was collected from YS Parmar University of Horticulture

and forestry, Nauni (Solan). P. euphratica leaf material was collected from TERI

field-station, Gual Pahari, Gurgaon, and P. maximowiczii leaf material was

kindly provided by Dr. Stefano Bisoffi of Poplar Research Institute, Italy.

3.1.1.2 Clonal Fidelity Testing

The study was carried out on P. deltoides G-48 clone micropropagated through

enhanced axillary branching in TERI. Twenty clones were selected and leaf

material was collected in three stages: 7th passage of micropropagation, 11th

passage of and from field transferred progeny.

3.1.1.3 Analysis of P. ciliata x maximowiczii hybrids

The study was carried out on 24 selected hybrid clones of P. ciliata X

maximowiczii, which were developed using P. maximowiczii pollen from Japan

and the two P. ciliata mother-trees, in the nursery of Dr. Y.S. Parmar University

of Horticulture and Forestry, Solan, India. The pollen were received from Oji

paper Company, Japan, but exact location of the source tree could not be traced

out, hence P. maximowiczii germplasm of known Japanese origin were included

in the study. The Japanese P. maximowiczii leaf material was kindly provided by

Dr. Stefano Bisoffi of Poplar Research Institute, Italy, and Dr. Pierre Perinet of

Ministère des Ressources naturelles du Québec , Sainte-Foy (Québec).

3

Materials & Methods


3.1.1.4 Development of Retrotransposon based Marker System

Many studies in plant genome analysis have shown the presence of high copy

number of retroelements e.g. maize and wheat. Keeping this in view,

retroelements were characterized to develop markers based on these sequences

in Populus. Three species were chosen for developing retro-transposon based

markers in Poplars: P. deltoides, P. ciliata and P. euphratica. More emphasis

was for the Himalayan poplar P. ciliata. The P. deltoides and P. ciliata leaf

material was collected from YS Parmar University of Horticulture and forestry,

Nauni (Solan). P.euphratica leaf material was collected from TERI field-station,

Gual Pahari, Gurgaon. The SSAP assay was tested for two types of analysis:

1. Genetic diversity study in P. deltoides accessions and

2. Hybridity testing of P. ciliata X maximowiczii hybrids.

3.1.1.5 Phylogenetic Analysis

Phylogenetic analysis based on 5S rDNA NTS was carried out on fifteen species.

The phylogenetic analysis of Populus species was carried out mainly with respect

to the species P. euphratica, whose leaf material was obtained from TERI field-

station, Gual Pahari, Gurgaon. Thirteen other Populus species were included in

the study namely P. alba, P. balsamifera, P. ciliata, P. deltoides, P. fremontii,

P. lasiocarpa, P. maximowiczii, P. nigra, P. schezuanica, P. simonii, P. tremula,

P. trichocarpa, and P. yunnanensis.

Salix tertasperma Roxb. was included as an ougroup in the study and its

leaf material was obtained from IHBT, Palampur.

3.1.2 Genomic DNA Isolation

All the experiments were performed after the first step of isolation of genomic

DNA. The CTAB method of Doyle and Doyle (1990) was used to isolate total

genomic DNA. All the solutions used in the experiments are listed in Annexure

II and the source of various chemicals and itms are given in Annexure III

1. 0.1 gm of lyophilized plant material was ground to fine powder in mortar

and pestle using quartz sand.

2. Powder was transferred immediately into 5ml of pre-warmed (65ºC)

isolation buffer, incubated for 30min at 65ºC in a water bath and mixed

gently every 10min.

3. Equal volume of chloroform : isoamylalcohol (24:1) was added and mixed.

The slurry was centrifuged at 10,000 rpm at 24ºC for 10 min. Aqueous

phase was transferred to fresh tubes.

Materials & Methods


4. 20 µl of RnaseA (10mg/ml) was added and incubated for 30 min at 37ºC.

5. Again equal volume of chloroform: isoamylalcohol was added, mixed gently

and centrifuged at 10000 rpm at 24ºC for 20 min. Upper aqueous phase was

transferred to glass centrifuge tubes using large bore pipette.

6. 0.6 volume of ice cold isopropanol was added, and mixed gently but

thoroughly by inverting the tubes several times. At this stage, the DNA -

CTAB complex was precipitated out. Then centrifuged at 6000rpm at 4ºCfor

10min. to pellet out the DNA.

7. The pellet was washed with washing solution (70% ethanol containing

10mM ammonium acetate) by gently agitating and collected by

centrifugation (10min, 5000rpm, 4ºC). The pellet was dried and 500µl of

M. Q. water were added to the DNA to dissolve overnight.

3.1.2.1 Purification of DNA

1. Phenol purification was used to purify all the crude DNA samples.

2. Equal volume of phenol: chloroform: isoamylalcohol (25:24:1) was added to

500µl of crude DNA prep.

3. The slurry was mixed gently with inversion and centrifuged for 15 min in

microcentrifuge Using a blunt / cut tip aqueous upper layer was taken out in

new eppendrof tubes.

4. Equal volume of chloroform isoamylalcohol was added to the aqueous layer.

It was mixed gently and centrifuged for 15 min.

5. Aqueous upper layer was taken out and 1/10th volume of 3M sodium acetate

(pH- 4.8) was added. It was mixed by inversion and incubated at 4ºC for

30min. A 2.5 volume of absolute alcohol was added, mixed by inversion and

incubated for half an hour at-20ºC. DNA was pelleted out by centrifugation.

6. The DNA pellets were washed with 70% alcohol for few minutes. The alcohol

was drained out on a paper towel and DNA was kept for drying at 37ºC.

7. To the DNA 200 µl of M.Q. water were added after proper drying and left

overnight at room temperature to dissolve.

8. All purified DNA were quantified on agarose gel by comparing with uncut

lambda DNA of known concentration.

3.1.2.2 Ammonium acetate purification

For further purification of the DNA samples, 0.5 volumes of 7.5M ammonium

acetate solution was added, and the samples were mixed and chilled on ice. The

samples were then centrifuged for 30 minutes (10,000 X g, 4 C) and the

supernatant was transferred to a clean tube. In order to precipitate the DNA,

two volumes of absolute ethanol was added to the samples and the contents

were mixed by inversion and stored for 1h at –20 C. The DNA pellet was

Materials & Methods


subsequently collected by centrifuging for 10 minutes at 5000 X g, 4 C, washed

in 70% ethanol and allowed to dry. The final pellet was dissolved in required

volume of ddw or TE buffer and stored at 4 or –20 C till further use.

3.1.2.3 DNA quantification

Qualitative and quantitative assessment of total genomic or plasmid DNA was

performed by subjecting the DNA samples to agarose gel electrophoresis

(Sambrook et al. 1989). Electrophoresis was carried out in 0.8% agarose gels in

0.5X TBE buffer at 30 mA for 2-3h (constant voltage equivalent to 3 Volts/cm)

to allow proper resolution. Subsequently, the gel was stained by ethidium

bromide (EtBr) at a final concentration of 0.5 g/ml and visualized under UV

light at short wavelength (254 nm) and photographed under red filter. Serial

dilution of uncut Lambda DNA (25-150 ng/ l) was used as standard molecular

size marker to quantify genomic DNA.

3.2 DNA Fingerprinting Methodologies

Four different DNA fingerprinting/profiling techniques were used in the present

research work namely ISSR, AFLP, SAMPL and SSAP techniques. The protocols

for each of these marker systems are given below.

3.2.1 ISSR assay

The PCR based marker system called Inter-Simple Sequence Repeat (ISSR)

access the variation present in the numerous microsatellite regions dispersed

throughout the various genomes (Figure 2.7, Chapter 2). Microsatellites are very

short (usually 10-20 base-pair) stretches of DNA that are “hypervariable",

present as different size variants within populations and among different

species. They are characterized by mono-, di- or tri- nucleotide repeats, e.g.,

(AA)n or (AG)n or (CAG)n etc, that have 4-10 repeat units present in tandem

(VNTRs). In ISSRs, one specifically targets the di- and tri- nucleotide repeat

types of microsatellite, because these are characteristic of the nuclear genome.

Inter-SSR Amplification or ISA is another terminology for ISSR. The assay is

usually carried out using anchored primer which ensure reproducibility and

gives more specificity to the assay.

The protocol followed for ISSR study was as follows:

1. 100 µg of the genomic DNA was taken for PCR amplification

2. PCR reaction mix was prepared as follows:

10X PCR Buffer 2.5 µl

25mM MgCl2 2.0 µl

Materials & Methods


10 mM dNTPs 0.5 µl

ISSR Primer 10µM 1.0 µl

Taq DNA polymerase (1U/µl) 0.5 µl

DD Water X µl

Total volume 25.0 µl

[10X PCR buffer (Fermentas): 750mM Tris-HCl (pH 8.8), 200mM

(NH4)2SO4, 0.1% Tween 20]

3. The following PCR conditions were applied for PCR:

94o C for 5 minutes Pre-amplification cycle

35 Cycles of the three steps of amplification:

94o C for 30 sec Denaturation

50o C for 45 sec Annealing

72 o C for 2 minutes Extension

72 o C for 5 minutes Post amplification extension

4o o C Soak temperature

4. On completion of the reaction the amplification products were separated

on 1.5% agarose gel and visualized using Ethidium bromide staining of

DNA in the gel and observing in UV light.

5. The bands were scored as 1 for presence and 0 for absence and data

matrix was obtained which was analyzed using the NTSYS pc software.

The sequences of the ISSR primers which were used in the present study are

given in Table 3.1.

3.2.2 AFLP assay

A schematic representation of AFLP methodology is provided in Figure 2.6

(Chapter 2). The AFLP analysis was performed as per the technical instructions

given by the manufacturers (Gibco, BRL). The sequences of the EcoR I and Mse

I specific adapters utilized in the present study are given as follows:

Adapters Sequence

Eco RI adapter

5'-CTCGTAGACTGCGTACC-3'

3'-CATCTGACGCATGGTTAA-5'

Mse I adapter 5'-GACGATGAGTCCTGAG-3'

3'-TACTCAGGACTCAT-5'

Materials & Methods


The steps involved in AFLP assay were:

1. Genomic DNA (250 ng) was restricted with both Eco R I and Mse I (1.25

U/ul each in a storage buffer comprising of 10 mM Tris-HCl, pH 7.4, 50

mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 50% BSA, 50%

glycerol, 0.1% Triton X-100) in a 5X restriction buffer (50 mM Tris-HCl,

pH 7.5, 50 mM Mg-acetate, 250 mM K-acetate) in a total reaction

volume of 25 l.

2. The restriction reaction was as follows:

5X reaction buffer 5 l

Genomic DNA (250 ng/ l) 18 l

Eco R I and Mse I enzyme mix 2 l

AFLP grade water to make up the final volume to 25 l

3. The contents were mixed gently and incubated for 2 h at 37 C.

Following the restriction, the enzymes were heat inactivated by

incubating the reaction mix for 15 minutes at 70 C.

4. Subsequently, Mse I and Eco R I adapters were ligated to digested

fragments in an adapter ligation solution in the presence of T4 DNA

ligase (1 U/ l).

5. The reaction was constituted as:

Restricted DNA (from previous step) 25 l

Adapter ligation solution 24 l

T4 DNA ligase (1U/ l) 1 l

Total Volume 50 l

6. The components of the reaction were mixed and incubated at 20 C for

2h.

7. The adapter ligated DNA was diluted 10 fold and taken for the pre-

amplification step.

8. Pre-amplification was carried out using primers corresponding to the

Eco RI and Mse I adapters with a single selective nucleotide at the 3’ end.

9. The sequence of the primers used are given in Table 3.2.

10. The reaction was constituted as:

Adapter ligated (diluted DNA samples) 5 l

Pre-amp primer mix 40 l

10X PCR Buffer (Gibco-BRL) 5 l

Taq DNA polymerase (1U/ul) 1 l

Total Volume 51 l

11. The reaction mix was centrifuged briefly after overlaying with 2-3 drops

of silicone oil.

Materials & Methods


12. The samples were pre-amplified using the following thermocycling

parameters: 20 cycles of : 30 s at 94 C; 60 s at 56 C; 60 s at 72 C

13. The pre-amplified DNA was diluted 50-fold and used as a template for

the selective amplification which employed +3N selective Eco R I and

Mse I primers. The sequences of the +3 selective primers are provided in

Table 3.2.

14. For the detection of the amplified AFLP fragments, Eco R I+3N primer

was end labeled. The reaction was assembled as follows:

Primers (Eco R I+3N) 18 l

5X Kinase Buffer (Gibco-BRL) 10 l

[ -32P] ATP (3000Ci/mmol) 20 l

T4 Polynucleotide Kinase (10 U/µl) 2 l

Total Volume 50 l

15. The reaction mix was incubated at 37 C for 1h. Following the labeling

reaction, the enzyme was heat inactivated at 70 C for 10 minutes.

16. For selective amplification, the reaction mix was assembled in two parts:

Mix I

Labeled Eco R I +3N primer 5 l

Mse I +3N primer (containing dNTPs) 45 l

Total Volume 50 l

Mix II

DD water 79 l

10X PCR buffer (Gibco-BRL) 20 l

Taq DNA polymerase (5 U/ l) 1 l

Total Volume 100 l

17. Mix I and Mix II were pooled together and 15 l of it was added to 5 l of

the template DNA (1:50 dilution of pre-amplified DNA).

18. Two to three drops of silicone oil was added to the reaction mix.

19. The cycling parameters for selective amplification were that of a touch

down PCR and were as follows:

One cycle of: 30 s at 94 C; 30 s at 65 C; 60 s at 72 C

Starting from 65 C, annealing temperature was lowered by 0.7

C at each subsequent cycle for 12 cycles in order to give a touch

down phase of 13 cycles

23 cycles of: 30 s at 94 C; 30 s at 56 C; 60 s at 72 C

20. The AFLP reaction was terminated by adding equal volumes of 98%

formamide dye.

21. The amplified products were electrophoresed in a 6% denaturing

polyacrylamide gel and autoradiographed (Sambrook et al. 1989).

Materials & Methods


Description on casting of polyacrylamide gels is provided after SAMPL

and SSAP protocols.

3.2.3 SAMPL assay

SAMPL protocol was based on AFLP assay with some modifications. A

schematic representation of the strategy employed for SAMPL analysis is

provided in Figure 2.9. Table 3.3 lists the sequences of the SAMPL primers

employed. The pre-amplified AFLP library was used for selective amplification

wherein an end labeled SAMPL primer was used in combination with a Mse I

+3N primer for PCR (see Table 3.2 for sequences of Mse I primer sequences).

The PCR conditions were same as that followed for AFLP except that the initial

/first annealing temperature in the thermocycling parameters for selective

amplification were modified based on the Tm of the SAMPL primer and Mse I+

3N primer utilized. The reaction for SAMPL analysis was terminated by adding

98% formamide dye. The amplified products were electrophoresed in a 6%

denaturing polyacrylamide gel and autoradiographed (Sambrook et al. 1989).

3.2.4 SSAP assay

SSAP technique is yet another modification based on the AFLP assay. A

schematic representation of the strategy employed for SSAP analysis is provided

in Figure 2.10. The pre-amplified AFLP library was used for selective

amplification wherein an end labeled SSAP primer was used in combination

with a Mse I +3N primer for PCR (see Table 3.2 for sequences of Mse I primers).

The SSAP primers were designed from retro-element sequences of Populus

deltoides (Dr. Wout Boerjan, personal communication). Primers of different

lengths and concentrations were tested for their utility as molecular marker for

Populus species. The primers were designed adjacent to the LTR regions and

their sequences were designated as PLTR-1 and PLTR-2 (Table 8.1, Chapter 8).

The PCR conditions were same as that followed for AFLP except that the initial

or first annealing temperature in the thermocycling parameters for selective

amplification were modified based on the Tm of the SSAP primer and Mse I+ 3N

primer utilized. The reaction for SSAP analysis was terminated by adding equal

volume 98% formamide dye. The amplified products were electrophoresed in a

6% denaturing polyacrylamide gel and autoradiographed (Sambrook et al.

1989).

Materials & Methods


3.3 Polyacrylamide gel electrophoresis

3.3.1 Preparation of gel plates

1. The glass plates were cleaned thoroughly with RO water and wiped dry

with lint free tissue paper. Following this, the plates were wiped dry

using absolute alcohol.

2. To prevent the gel from sticking to the plates, the thermostatic plates

were treated with 5 ml repel-silane (2% diethyl di-chloro silane in 1, 1, 1,

1, tri-chloroethane). Repel silane was evenly spread on the glass plate

and polished dry with lint free tissue paper.

3. The procedure was repeated twice and the plate was finally cleaned with

absolute alcohol.

3.3.2 Assembly of the casting apparatus

1. The thermostatic plate was placed over the casting stand horizontally

and the level was balanced with an oil drop.

2. Spacers of 0.4 mm thickness were secured firmly on the thermostatic

plate with the help of binding clips taking care that the spacers don't

form loops.

3. The notched side of the glass plate was gently placed on the thermostatic

plate in such a manner that the top portion of the notched plate was

juxtaposed against the bottom of thermostatic plate.

3.3.3 Preparation of polyacrylamide gel

To prepare PAGE, the following components were added:

Components For 6% gel

Urea 21 g in 20 ml ddw

40% acrylamide 7.5 ml

20X TBE 2.5 ml

Final volume 50 ml

1. For polymerizing the gel, 500 l of 10% of freshly prepared ammonium

persulphate and 50 l of TEMED was added just before casting.

2. The gel solution was poured between the two plates and the notched

plate was gently slided over the thermostatic plate. Care was taken to

prevent any air bubble from getting trapped.

3. A 0.4 mm square toothed comb was inserted to form wells and the two

plates were tightly secured with the help of clamps and clips.

4. The gel was allowed to polymerize for few hours.

Materials & Methods


5. Prior to loading, the wells were cleaned by flushing with 1X TBE with the

help of a syringe.

6. The glass plate assembly was connected to power-pack at a constant

voltage supply of 2000 V and bath circulator to maintain the

temperature at 55 C.

7. The gel was pre-run at 30V/cm for 30 minutes to remove the charged

particles and to bring the gel to 55 C.

3.3.4 Sample loading

1. An equal volume of 98% formamide dye was added to the samples and

the samples were heat denatured at 100 C for 5 minutes.

2. Two to 3 µl of denatured samples were loaded per well.

3. The samples were allowed to run till xylene cyanol (corresponding to 50

bp) was about 4 inches above the lower end of the gel.

3.3.5 Gel drying and autoradiography

1. The gel was vacuum dried at 80 C for 1h and exposed overnight to X-

Ray film at –80 C.

2. The exposed X-ray films were developed and fixed (Sambrook et al.

1989).

3.4 Statistical analysis of fingerprinting data

Phenetic relationships were interpreted for the molecular marker data using

statistical software package NTSYS-pc software (version 2.02, 1994)

3.4.1 Construction of binary matrix by fragment matching

The molecular data was scored for presence (1) and absence (0) of bands across

the genotypes to construct a binary matrix. Co-migrating bands were assumed to

represent the same locus. Only clearly amplified bands were scored. Ambiguous

bands with faint amplification signals were omitted from the analysis.

3.4.2 Calculation of similarity matrix

The resultant matrices were statistically analyzed using NTSYSpc software,

version 2.02 (Rohlf 1998). Two different data sets were used for the analyses in

this study: (1) total scorable bands (total bands), and (2) polymorphic bands

only (i.e. total bands minus monomorphic bands). The data matrix was used to

calculate genetic similarity between accessions i and j using three different

coefficients namely- Nei and Li (1979) [GSNL = 2a/(2a+b+c)], Sokal and

Michener (1958) [GSSM =(a+d)/(a+b+c+d)] and Jaccard (1908) [GSJ=

a/(a+b+c)], where a is the number of bands shared by i and j, b is the number of

Materials & Methods


bands present in i and absent in j, c is the number of bands present in j and

absent in i, d is the number of bands absent in i and j, and (a+d) is the number

of bands matched (number of bands present and absent in i and j) for the data

pooled over all the primer combinations.

3.4.3 Cluster analysis and construction of

phenetic dendrogram

The similarity matrix was subjected to Sequential Agglomerative Heirarchial

Nested Clustering (SAHN). The resulting distance matrices were subjected to

four clustering methods: UPGMA (unweighted pair group method analysis;

Sokal and Michener 1958), WPGMA (weighted pair group method analysis;

Sneath and Sokal 1973), complete linkage (Lance and Williams 1967), and single

linkage (Lance and Williams 1967) in order to group individuals into discrete

clusters. The cophenetic correlation coefficient was calculated to test the

goodness of fit between the similarity matrix and the cophenetic matrices

(Sneath and Sokal 1973).

3.4.4 Principal Component Analysis

Principal Component Analysis plots allows us to visualize the genetic

similarities/differences as a multi-dimensional solution of the observed

relationships. This method resolves complex relationships into interactions of

fewer simpler factors. Presence and absence of fragments in OTUs are used to

plot a hyperellipsoid in a multi-dimensional space. To simplify the description of

these clouds of points, the principal axes through the hyperellipsoid are

calculated. The 1st major axis accounts for the highest amount of variation. The

2nd principal axis explains the second highest amount of variation. Thus, the

successive principal axes account for greatest and next greatest amount of

variation (adapted from Weising et al. 1995). Principal Component Analysis was

carried out on the similarity matrix, with the scores on the resultant principal

axes (1st two) plotted pairwise.

3.4.5 Mantel test

Mantel test (Mantel, 1967) was used to test the significance of the correlation

coefficient between pairs of similarity matrices and for determining cophenetic

correlation values. Mantel test provides the values for "goodness of fit".

According to Rohlf (1998), the value for coefficient of correlation (r), which is

greater than 0.85, suggests a goodness of fit.

3.4.6 Bootstrap analysis

Materials & Methods


The bootstrap values for the degree of confidence at the nodes of dendrograms

were analyzed by Bootstrap Analysis (Felsenstein et al. 1985) using WINBOOT

software (Yap and Nelson 1995). Bootstrap values were calculated by

constructing phenetic dendrograms 100-1000 times and determining the

support values for each node as a percentage. Each dendrogram was derived

from a distinct, modified binary matrix, which was different from the original

binary matrix. From the original binary matrix, few loci were randomly omitted.

The deleted loci were substituted by loci derived from the remaining binary

matrix. Hence, an infinite number of binary matrices could be generated from

the original matrix by utilizing different permutations and combinations. This

way, 100 to 1000 phenetic dendrograms could be constructed for calculation of

bootstrap values.

3.4.7 Polymorphism Information Content (PIC)

PIC or Average heterozygosity, Hav, maybe calculated using the following

formula (Powell et al. 1996):

Hav = Hp/np

Where Hav is the average heterozygosity calculated from all the bands

(monomorphic + polymorphic bands), is the proportion of polymorphic bands

i.e. Polymorphic bands/Total number of bands, Hp =1-Pi2 where Pi is the

proportion of genotypes having the ith allele, np is the number of polymorphic

bands. The resultant value is averaged by dividing it with total number of assays

or primer combinations employed.

3.5 Isolation and cloning of 5S rDNA spacer

The tandem repeat DNA sequences as exemplified by 5S rDNA are located in

one or more chromosomal loci in both the higher plants and animals. 5S rDNA

repeat unit comprises of a 120 bp coding region which is separated from the next

coding sequence by an intervening Non-Transcribed-Spacer (NTS) which, in

higher plants, is known to vary in length from 100 to 700 bp. The sequence and

length of 5S rDNA coding region is highly conserved whereas, the NTS varies

from species to species. The primers "A" and "B" were designed in such a

manner that they annealed to conserved sequences of 120 bp coding region at

nucleotides 69-88 and 28-49 and resulted in the amplification of the entire NTS

stretch with 5' and 3' flanking sequences corresponding to the preceding and

following coding region, respectively (Figure 3.1). The primers were based on the

120 bp coding region of Brassica juncea (Bhatia et al. 1993). 5S rDNA NTS

regions were cloned as amplification products from various Populus species

according to the strategy schematically represented in Figure 3.1.

Materials & Methods


3.5.1 PCR amplification of 5S rDNA non-

transcribed spacer

The 5S rDNA NTS (spacer region) was amplified from the whole genomic DNA

using Primer A and Primer B, designed complementary to the conserved region

of 5S gene.

Sequence of Primers:

Primer "A": 5'- TGCGATCATACCAGCACTAA-3'

Primer "B": 5'- GGGAAGTCCTCGATGTTGCA-3'

1. PCR reactions were prepared in a total reaction volume of 50.0 l as

follows:

Components for the reaction Volume per reaction

DNA (25-50 ng) 10.0 l

10X PCR Buffer 5.0 l

25 mM MgCl2 5.0 l

Primer"A"(20 pmol/ l) 2.0 l

Primer"B" (20pmol/ l) 2.0 l

10 mM dNTP 1.0 l

Taq DNA polymerase (5U/ l) 0.5 l

DDW 24.5 l

Total Volume 50.0 l


(NH4)2SO4, 0.1% Tween 20]

2. The reaction mix was overlaid with a drop of mineral oil and placed in a

thermal cycler. An initial denaturing step was given at 94 C for 5 minute

followed by a PCR profile of:

Denaturing step of 94 C for 45sec

Annealing step of 55 C for 45sec

Extension step of 72 C for 1 minute

Total cycles: 35

Final extension step of 72 C for 7mins; (4 C soak temperature).

3. After the completion of the reaction, an aliquot of the product was loaded

in 1.2% agarose gel along with appropriate molecular size marker and

electrophoresed to confirm the amplification.

4. The putative NTS was subsequently "A" tailed, purified and cloned in a

p-GEMT vector (Annexure IV).

5. Finally, the 5S rDNA spacers were characterized by Southern blot and

sequence analysis.

Materials & Methods


3.5.2 Purification of PCR amplification products Wizard PCR Preps DNA purification system (Promega)

The PCR products were purified prior to cloning to eliminate contaminants

including primers, primer-dimers, salts and other macro-molecular substances.

For each PCR product, the aqueous phase was transferred to a micro-centrifuge

tube to get rid of mineral oil as much as possible. The following steps were

performed as per the Wizard PCR Preps DNA purification system kit

instructions.

1. Direct Purification Buffer (100 l) was aliquoted into a 1.5 ml micro-

centrifuge tube.

2. 30 to 300 l for PCR reaction was added and vortexed briefly to mix.

3. One ml of the resin was added and the mixture was vortexed briefly three

times over a one-minute period.

4. For each PCR product, one mini-column (supplied with the purification

system) was prepared.

5. The resin/DNA mix was added into the syringe barrel. The syringe plunger

was inserted slowly and the slurry was gently pushed into the mini-

column.

6. The syringe barrel was detached from the mini-column and the plunger

removed.

7. The barrel was reattached to the column and 2 ml of 80% isopropanol was

pipetted into the syringe barrel.

8. The syringe plunger was inserted into the syringe and gently pushed to

wash the mini-column.

9. The syringe was removed and the mini-column was transferred to a micro-

centrifuge tube. It was centrifuged for 2 minutes at 10,000 X g to dry the

resin.

10. The mini-column was transferred to a new micro-centrifuge tube and 50 l

of water was applied.

11. After 1 minute, the mini-column was centrifuged for 20 s at 10,000 X g to

elute the DNA fragment.The yield of the eluted product was checked on

agarose gel.

3.5.3 “A" tailing of PCR amplified product

After confirming the amplification of NTS on the agarose gel PCR products were

A-tailed to facilitate cloning in the pGEM-T vector. (For details on pGEM-T

vector system see Annexure IV.

1. PCR product was directly A- tailed by adding dATP to a final

concentration of 0.2 mM and Taq polymerase in the following reaction:

Materials & Methods


PCR product (~100ng) 25.0 l

10X PCR buffer 1.5 l

50mM MgCl2 1.5 l

2 mM dATP 1.5 l

Taq polymerase5U/ l) 0.2 l

Total Volume 15.0 l

[10X Reaction Buffer (Promega): 100mM Tris-HCl (pH 9.0 at 25°C),

500mM KCl, 1% Triton® X-100]

2. "A" tailing was accomplished by incubating the reaction at 72 C for 15

minutes.

3. Thereafter the A-tailed products were ligated to pGEM-T vector and

transformed into E.coli cells.

3.5.4 Ligation of A-tailed PCR products

1. Purified PCR reactions (5S rDNA NTS) were ligated into pGEM-T

cloning vector system of Promega (for details on pGEM-T vector system

see Annexure IV)

2. Briefly centrifuged the pGEM-T vector and purified PCR reactions to

collect contents at the bottom.

3. Ligation reactions were set up as follows:

2X Rapid Ligation Buffer(kit) 10.0µl

pGEM-T vector 1.o µl

PCR product 8.0µl

T4 DNA Ligase 1.0µl

Total Volume 20.0 l

Vortexing the 2X Rapid Ligation Buffer vigorously essential

before use.

4. Mixed the reactions by pipetting. Incubate the reactions overnight at

16 C.

5. Heat inactivated the enzyme (T4 DNA Ligase) by incubating at 70 C for

10 minutes.

6. The reaction was used for transformation into E.coli cells as described in

the next section.

Materials & Methods


3.5.4 Transformation

3.5.5.1 Preparation of competent bacterial cells

Escherichia coli strains (NM522, DH5 , XL-1 Blue) were selected for

transformation. Competent cells were prepared following the CaCl2 method as

described by Mandel and Higa (1970).

1. The host strain was revived from 30% glycerol stocks by streaking on L-

Broth (Agar) [LB-Agar: Tryptone, 10 g; Yeast Extract, 5 g; NaCl, 10 g;

Agar, 15 g; dH2O, 1 L]

2. A single colony from the isolated colonies, was inoculated in 2 ml of L-

Broth the next day and grown overnight at 37 C at 200 rpm.

3. 500 l of overnight culture was inoculated into 50 ml of fresh L-Broth

the next day.

4. The bacteria were allowed to grow at 37 C and 200 rpm till the optical

density at 585 nm was 0.3.

5. The culture was chilled on ice for 10 minutes and the cells were

harvested by centrifugation at 4000 X g for 10 minutes at 4 C.

6. The pellet was gently suspended in 10 ml of chilled 0.1 M CaCl2 and

incubated on ice for 20 minutes.

7. The cell suspension was centrifuged at 4000 X g, 10 minutes and the

pellet was gently re-suspended in 1 ml of 0.1 M chilled CaCl2 using a

wide bore pipette.

8. The competent cells thus prepared were left on ice till further use.

3.5.5.2 Transformation of competent cells

For transformation of the competent cells the following steps were performed:

1. 10 µl of ligation mix or supercoiled undigested plasmid DNA (1 ng to

determine the efficiency) were added to 250µl of competent cells and

incubated on ice for 30 minutes.

2. A brief heat shock for 2 minutes was given by incubating in water-bath

set at 42 C.

3. The reaction was transferred immediately transferred to ice and

incubated for 2 minutes.

4. One ml of L-Broth was added to the transformation mix and the cells

were allowed to recover and grow at 37 C, 1h at 200 rpm.

5. A flash spin (10,000 X g, RT, 2 minutes) was given to the transformation

mix and the transformed cells were obtained as a pellet.

6. The cells were re-suspended in 100µl of LB-media and plated onto LB-

agar IXA plates containing IPTG (50 µg/ml), X-Gal (50 µg/ml) and

Ampicillin (100 µg/ml).

Materials & Methods


7. The transformed cells were allowed to grow selectively on the antibiotic-

containing medium overnight at 37 C.

8. White colonies containing the putative recombinants were observed after

overnight incubation and re-gridded on duplicate plates.

3.5.6 Screening of the positive recombinants by colony PCR

1. PCR amplification of the cloned inserts was carried out on colonies

(colony PCR).

2. A part of colony was picked up with sterile tooth prick and re-suspended

in the PCR reaction mix.

3. Few colonies were selected at random and the insert was PCR amplified

to check for the size of insert.

4. The reaction was assembled as follows:

10X PCR Buffer 1.5 µl

25 mM MgCl2 1.5 µl

10 mM dNTPs 0.3 µl

Primer T7/Forward Primer (20 pmol/µl) 0.6 µl

Primer SP6/Reverse Primer (20 pmol/µl) 0.6 µl

Taq polymerase (5 U/µl) 0.1 µl

ddw 11.0 µl

Total Volume 15.0 µl


(NH4)2SO4, 0.1% Tween 20]


thermal-cycler.

6. An initial denaturing step was given at 94 C for 1 minute followed by the

following steps:Denaturing step of 94 C for 45 sec; Annealing step of

50 C for 45 sec; Extension step of 72 C for 1 min for a Total of 35 cycles

7. Final extension step of 72 C for 7mins, followed by 4 C soak

temperature.

8. For screening those colonies in which the desired insert has been cloned,

colony PCR reactions were ectrophoresed on agarose gel.

9. Then under U.V light each amplified product of ~500bp was recorded for

its absence or presence.

10. The positive clones were grown in 2 ml cultures (L-Broth, Ampicillin 100

µg/ml) overnight and plasmids were isolated.

Materials & Methods


The sequences of the universal primers utilized are as follows:

Primer Sequence

SP6 (19mer) 5'-TATTTAGGTGACACTATAG-3'

T7 (20mer) 5'-TAATACGACTCACTATAGGG-3'

3.5.7 Plasmid DNA isolation

Plasmid from all the colonies which showed presence of desired insert (5S

rDNA) of ~500bp was isolated. Plasmids were isolated using various protocols

depending upon amount obtained. Alkaline lysis method of plasmid purification

(mini prep) was used for making probe and PCR amplification was carried out

on plasmid DNA for size-estimation of the insert. For sequencing, the plasmids

were subjected to alkaline lysis method with optional PEG-NaCl purification

steps or commercially available columns were used.

3.5.7.1 Alkali-lysis method for isolation of plasmid DNA

The Compositions of the Solutions used are given in annexure II

1. Bacterial cells containing recombinant plasmids were grown overnight at

37 C in a 2 ml culture (LB medium with ampicillin).

2. The cells were pelleted down by centrifugatiom for 30 seconds and the

supernatant was discarded.

3. The pellet was resuspended by vortexing in 100 µl of ice cold "Solution I"

and was incubated for 5 minutes at room temperature.

4. "Solution II" (200 µl) was added and the contents were mixed by

inversion. This was followed by incubation on ice for 5 minutes.

5. Finally, 150 µl of ice cold "Solution III" was added and mixed by

inversion followed by further incubation on ice for 5 minutes.

6. The suspension was centrifuged for 10,000 X g for 15 minutes and the

supernatant was collected.

7. RNAse A was added to the supernatant to a final concentration of 20

µg/ml and was incubated at 37 C for 30 minutes.

8. The mixture was extracted with equal volume of phenol: chloroform:

isoamyl alcohol (25:24:1, v/v) and centrifuged for 15 minutes to separate

the phases.

9. The aqueous phase was transferred to a fresh tube and re-extracted with

equal volume of chloroform: isoamyl alcohol (24:1).

10. To the aqueous phase, 1/10th volume of sodium acetate (pH 4.8) was

added followed by 2.5 volumes of absolute alcohol.

11. DNA was precipitated at –20 C for 30 minutes and then pelleted by

centrifugation.

Materials & Methods


12. The pellet was either dissolved in 20 µl of ddw or further purified by PEG

method.

13. For PEG purification, the pellet was dissolved in 16 µl ddw. Following

this, 4 µl of 4 M NaCl was added.

14. After thoroughly mixing the contents, 20 µl of 13% PEG (w/v) was added

and the samples incubated on ice for 20 minutes.

15. The samples were centrifuged for 10 minutes to remove the supernatant

and the pellet washed with 1ml pre-chilled 70% ethanol followed by

absolute alcohol.

16. The pellet was air dried and dissolved in desired amount of double

distilled water or TE buffer.

3.5.7.2 Wizard TM Plus miniprep DNA purification

The Promega Wizard TM Plus miniprep DNA purification system was used for

isolating plasmid DNA for sequencing purpose. The Kit supplied all the

reagents. The following steps were performed according to the manufacturer’s

instruction:

1. 2 ml of overnight (16h) bacterial culture of DH5 / NM522 transformed

with recombinant plasmids were utilized for plasmid DNA isolation.

2. The cells were pelleted by centrifuging 2 ml of culture for 30 seconds at

10,000 X g at 4 C.

3. The cell pellet was resuspended in 200 µl of "Cell Resuspension"

solution.

4. Two hundred micro-litres of "Cell Lysis" solution was added and the

contents mixed by inverting the tube 4-5 times.

5. Neutralizing solution was added and the reaction mixed by inverting the

tube 4-5 times.

6. The lysate was centrifuged at 10,000 X g in a micro-centrifuge for 5

minutes.

7. One WizardTM minicolumn was prepared for each miniprep. The plunger

was removed from 3 ml disposable syringe and set aside.

8. The syringe barrel was attached to the Luer-Lok extension of the

minicolumn and 1ml of resin was re-suspended into the barrel.

9. All of the cleared lysate was carefully removed from each miniprep and

transferred to the barrel of the minicolumn/syringe assembly containing

the resin.

10. The syringe plunger was carefully inserted and the slurry gently pushed

into the minicolumn.

11. The syringe was detached from the minicolumn and the plunger

removed from syringe barrel.

Materials & Methods


12. The syringe barrel was to reattached the minicolumn. Two ml of

"Column Wash" solution was pipetted into the barrel of the

minicolumn/syringe assembly.

13. The plunger was inserted into the syringe and the "Column Wash"

solution gently pushed through the minicolumn.

14. The syringe was removed and the minicolumn transferred to a 1.5 ml

micro-centrifuge tube and centrifuged at 10,000 X g for 2 minutes to dry

the resin.

15. The minicolumn was transferred to a new 1.5 ml micro-centrifuge tube.

16. 50 µl of water was applied and waited for 1 minute.

17. The column was centrifuged for 20 s at 10,000 X g to elute the plasmid

DNA.

18. The isolated plasmid DNA was checked on 1% agarose gel by ethidium

bromide staining.

3.5.8 Size-estimation of the inserts

1. PCR was carried out to check the size of the inserts in the plasmid using

the following reaction:

Plasmid DNA (20-50ng) 2.0 µl

10 X PCR Buffer 2.0 µl

25 mM MgCl2 2.0 µl

10 mM dNTPs 0.3 µl

SP6 Primer (20 µM) 0.3 µl

T7 Primer (20 µM) 0.3 µl

DDW 9.9 µl

Taq DNA polymerase (1 U/µl) 0.2 µl



(NH4)2SO4, 0.1% Tween 20]


thermal cycler. The cycling parameters followed were:

1 cycle of: 4 minutes at 94 C; 2 minutes at 50 C; 2 minutes at 72 C

25 cycles of: 1 minute at 94 C; 1 minute at 50 C; 2 minute at 72 C

Final Extension step of 72 C for7 minute (4 C soak temperature).

3. The amplified products were resolved on a 1.2 % agarose gel along with

1Kb ladder.

Materials & Methods


3.6 Southern analysis for genome organization

of 5s rDNA sequences

3.6.1 Restriction of genomic DNA

All restriction endonuclease digestions were carried out as per the

manufacturer’s instructions. Two to five µg of genomic DNA was taken for

restriction and three to five units of enzyme was used per µg of DNA. One µl of

RNase A was added to remove the traces of RNA and spermidine (1 µl) was

added to aid in the digestion. Bam HI was chosen for southern analysis as the

conserved region of the 5S rDNA is known to contain the recognition site

(GGATCC) for this enzyme.

1. The restriction reaction was set up as follows:

Genomic DNA (2-5 µg) X µl

10X Restriction Buffer 3 µl

RNAse A (10 mg/ml) 1 µl

Spermidine (10 µg/ml) 1 µl

Bam HI Enzyme (20 U/µl) 3-5 U/µg of DNA

DDW Y µl for total


[The restriction buffer was as provided by the supplied]

2. Restriction of genomic DNA was carried out for 16 to 18h at 37oC for

complete restriction.

3.6.2 Southern blotting

3.6.2.1 Gel treatment

DNA samples were run overnight in 0.8 to 1.0 % agarose gels, 0.5X TBE buffer

at 15V (constant voltage). The gel was then surface stained and photographed.

The fractionated DNA was transferred on to nylon/ nitro-cellulose membrane by

capillary blotting technique. Prior to Southern blotting, the following treatments

were given to the fractionated DNA in the gel with constant but gentle shaking to

facilitate efficient transfer.

1. Depurination: The gel carrying the fractionated DNA was submerged in

0.25 N HCl for 20 minutes at room temperature or till ten minutes after

the colour of the blue dye changed to yellow.

2. Denaturation: After depurination, the gel was treated with Denaturation

solution [0.5M NaoH, 1M NaCl] to denature the double-stranded DNA

molecules to single stranded molecules. This step was carried out for 40

minutes at room temperature.

Materials & Methods


3. Neutralization: The denatured DNA was neutralized in neutralization

buffer [0.5M Tris HCl (pH 7.4), 3.0M NaCl] for 1h at room temperature.

After each step, the gel was rinsed with RO water.

3.6.2.2 Assembly of capillary blot

1) For capillary transfer of DNA to nylon membrane, the procedure as

described by Sambrook et al. (1989) was followed.

2) A capillary stand was placed on a tray filled with 20X SSC which was the

transfer medium.

3) A sheet of Whatmann 3MM paper was used as a wick.

4) Three sheets of Whatmann 3MM paper were cut to the size of the gel and

soaked in 20X SSC. These were placed on the wick.

5) The gel was inverted and placed on the Whatmann sheets taking care

that no bubbles were trapped between the Whatmann sheets and the gel.

6) Nylon membrane was cut to the size of the gel, soaked in 20X SSC and

placed carefully over the gel, avoiding the entry of the bubbles between

the gel and the nylon membrane.

7) On the top of the membrane, three sheets of Whatmann 1MM, cut to the

size of the membrane and pre-wet with 20X SSC were successively

layered again taking care that to avoid air bubbles.

8) A stack of blotting sheets was placed on the Whatmann sheets and a

weight of 500 gm was placed on the stack.

9) Capillary transfer was allowed to proceed for 20h.

10) After complete transfer, the blotting sheets and filter papers were

carefully removed and the gel along with the nylon membrane was

placed on a fresh blotting sheet with gel side up.

11) Orientation and the position of the wells were marked on the nylon

membrane.

12) The membrane was air-dried.

3.6.2.3 UV Crosslinking

The DNA transferred to the nylon membrane was covalently fixed to the

membrane by UV crosslinking at 1.2 x 105 µJ/cm2. Complete transfer of the DNA

to the nylon membrane was ensured by re-staining the gel after Southern

transfer and visualization. Following this, the detection of the signal was effected

by using radioactive method.

Materials & Methods


3.6.3 Southern hybridization & signal detection

3.6.3.1 Probe labelling by random priming method

Radioactive labelling of the probe was carried out using random hexamer

primers (Feinberg and Vogelstein 1983). The 5S rDNA NTS was PCR amplified

from the plasmid containing the clone and taken as probe for the analysis.

Double stranded DNA (25 ng) was taken in 10 µl volume and heat denatured

thoroughly in a water bath (100 C) for 5 minutes followed by chilling on ice.

1. Reaction mix: PCR pr0duct (25ng) 10 µl

dATP (500 M) 1 µl

dGTP (500 M) 1 µl

dTTP (500 M) 1 µl

2.5 X (Buffer) 20 µl

-32 P-dCTP (3000Ci/ mmol) 5 µl

DNA Pol I (Klenow fragment) 1U/µl 2 µl

DDW (to make up the final volume to 50 µl) x µl

Final Volume 50 µl

2. The reaction mixture was incubated at 37 C for 30 minutes.

3. The reaction was stopped by addition of 5 µl of 0.5 m EDTA, pH 8.0.

3.6.3.2 Prehybridization

The protocol for prehybridization and hybridization was used as given by

Lakshmikumaran et al. (1985). The UV-crosslinked nylon membrane was

prehybridized in the pre-hybridization buffer at 55–60 C for 16h (over-night).

The whole experiment was set up in a radio-active shield containing rotisserie

shaker in a room maintained for radioactivity work.

3.6.3.3 Hybridization of radio-labeled probe

1. Addition of radiolabelled probe: The labelled probe was added to the

bottles containing the membranes in the pre-hybridization buffer.

Hybridization of the probe to the target DNA was carried out under high

stringency conditions.

2. High stringency hybridization and washing: The nylon membrane was

pre-hybridized at 60–65 C for at least 16h. Subsequently, the buffer

containing unbound probe was properly discarded and the membrane

was given two washes of 2X SSC + 0.1% SDS at room temperature or at

60 C for 20 minutes each followed by a wash with 3 mM Tris-Base at

room temperature for 20 minutes.

Materials & Methods


3.6.3.4 Signal detection by autoradiography

The nylon membrane hybridized to total genomic DNA was exposed to an X-ray

film and autoradiographed at –80 C overnight. Hyper-screen coated with rare

earth metal was placed in the cassette in order to amplify the signal intensity.

The film was subsequently developed using standard procedure as shown by

Sambrook et al. 1989.

3.7 Sequencing and characterization of the

5S rDNA non coding spacer

Sequencing of double stranded DNA was based on the di-deoxy chain

termination method of Sanger et al. (1977). For automated sequencing, ABI

PRISM 310 genetic analyzer automated sequencer platform was utilized. The

cycle sequencing reactions were carried out using ABI PRISM Big Dye

Terminator cycle sequencing Ready Reaction Kit, v2.0.

3.7.1 Terminator Ready Reaction for cycle

sequencing

Column purified plasmids were quantified and the reaction was assembled as

follows:

Terminator ready reaction mix 8.0µl

Template DNA (plasmids, 200ng to 500ng) 8.0µl

T7 Primer (3.2 pmol) 1.0µl

ddw 3.0µl

Total volume 20µl

The components of ready reaction mix were as follows:

-A-Dye terminator labeled with dichloro [R6G]

-B-Dye terminator labeled with dichloro [ROX]

-C-Dye terminator labeled with dichloro [R110]

-D-Dye terminator labeled with dichloro [TAMARA]

Deoxynucleoside triphosphates (dATP, dCTP, dITP, dUTP)

AmpliTaq DNA polymerases, FS

MgCl2

Tris HCl Buffer, pH-9.0

The samples were mixed thoroughly, overlaid with 40 ul of mineral oil.

Following this, the reaction was cycle-sequenced under the following

thermocycling parameters:

Materials & Methods


25 Cycles of:

Rapid thermal ramp (1 C/s) to 96 C

30 s at 96 C


15 s at 50 C


4 min at 60 C

Finally, a rapid thermal ramp was achieved to 4 C.

The reaction mix was allowed to remain at 4 C till further use.

3.7.2 Purification of extension products by ethanol/sodium acetate precipitation

Two microlitre (corresponding to 1/10th volume) of 3M sodium acetate (pH 4.6)

was added to the reaction mix. Fifty µl of 95% ethanol (corresponding to 2.5

volumes) was subsequently added to the samples and the tubes were inverted

several times to allow a thorough mixing. The extension products were allowed

to precipitate for 15 minutes at room temperature. The pellet was washed with

70% alcohol and allowed to dry.

3.7.3 Electrophoresis on ABI PRISM 310 genetic analyzer

Since the inserts to be sequenced were short in length, we used a capillary that

was 47 cm long and 50 µm wide. The capillary was coated with POP-6TM with the

help of 1 ml syringe and the running module for carrying out this step was Seq

POP 6 (1 ml) Rapid E run module. Finally, the samples were loaded and allowed

to run for 36 minutes. The sequences were obtained as electropherograms.

3.8 Sequence analysis of 5S rDNA spacers

Sequence analysis for determining restriction enzyme site was done by the help

of the software Webcutter 2.0. It is available free thorough the website

<http://www. ccsi. com/ firstmarket/ firstmarket/ cutter/ cut2. html>.

The software Sequence Massager was utilized for proper aligning of the

sequences. ClustalW program (Thomson et al. 1994, http://www.ebi2.ac.uk/)

was used for aligning the 5s rDNA nontranscribed spacer sequences based on

the overall similarities and for obtaining a cladogram based on sequence data.

The sequence alignment was also scrutinized and manually aligned to get best

alignment. PAUP* (Swofford 1991), TREEVIEW (Page 1996), DNAsp (Rozas et

al. 2003), MEGA (Kumar 1994) softwares were used for detailed analysis which

are described in Chapter 7 and 8.

Materials & Methods


3.9 Isolation and cloning of Copia-RT region

3.9.1 PCR amplification and cloning of Reverse

Transcriptase (RT) sequences

The partial Reverse Transcriptase (RT) sequences were amplified from P. ciliata

and P. euphratica genomic DNA using the degenerate primers from the RT

regions which are highly conserved in the plants for the Copia retroelements

(Flavell et al. 1992a).

The primers sequences were designed from the conserved motifs and were

commercially synthesized on order. The sequences of the primers are:

RT-F: 5’-ACNGCNTT(C/T)(C/T)TNCA(C/T)GG-3’

RT-R: 5’-A(A/G)CAT(A/G)TC(A/G)TCNAC(A/G)TA-3’.

1. PCR reactions was prepared as follows:

25-50 ng of genomic DNA

1X PCR buffer

2.0 mM of MgCl2

0.2 mM dNTP

1.0U of Taq DNA polymerase

0.4 M of the primers RT-F

0.4 M of the primers RT-R

Total reaction volume = 50.0 l.


(NH4)2SO4, 0.1% Tween 20]

2. PCR parameters were:

94 C for 5 minute

Denaturation at 94 C for 45sec; annealing at 50 C for 45sec; extension

at 72 C for 1 minute X30 cycles

Final extension at 72 C for 7mins.

3. Amplification products were purified with the WizardTM purification system

from Promega and were ligated into pGEM-T cloning vector.

4. The recombinant plasmids were transformed into Escherichia coli DH5-α or

JM109 supercompetent cells (Promega) by electroporation technique.

Materials & Methods


3.9.2 Transformation by electroporation

The electropotation procedure was employed due to its high efficiency of

transformation

3.9.2.1 Preparation of Electro-competent cells

1. A single colony of E.coli (DH5 ) was obtained by streaking a 30% glycerol

culture (stored in –80 0C) on Luria Broth (LB) Agar. This was used to

inoculate 5ml LB and was grown at 370C, 225rpm overnight.

2. 12.5ml of this overnight culture was used to inoculate 250ml of LB in 1 litre

flask. The culture was grown for 2-2.5hrs at 370C, 300 rpm until optical

density of the suspension reached upto 0.6.

3. The cells were snap cooled on wet ice for 20mins, were poured into chilled

50ml centrifuge bottles and centrifuged at 4000xg for 15mins at 40C.

4. The supernatant was decanted off and the pellet was resuspended in 250ml

of ice-cold 10% glycerol.

5. The suspension was centrifuged for 15mins at 4000xg, 40C.

6. The supernatant was decanted off and the pellet was resuspended in 125ml

of ice-cold 10% glycerol.


8. The supernatant was decanted off and the pellet was resuspended in 10ml of

ice-cold 10% glycerol.


10. The supernatant was decanted off and the pellet was resuspended in 1ml of

ice-cold 10% glycerol.

11. Finally, 50 l of these electrocompetent cells were flash cooled in liquid

nitrogen and stored in –700C until transformation.

3.9.2.2 Electroporation

1. Frozen aliquots of electrocompetent cells were thawed on ice.

2. A 0.2cm electroporation cuvette (Gap 5; BioRad) was super chilled by

placing it in –20 0C for 1-2hrs.

3. The thawed electrocompetent cells (40 l) were mixed with the ligation

mix (purified and dissolved in 4 l sterile water) and incubated in ice for

1min.

4. The mixture of cells and DNA were transferred to the ice-cold cuvette

and tapped until the solution reached the bottom of the cuvette.

5. After wiping the surface of the cuvette dry, it was placed in the chamber

slide of the electroporator and pushed between the contacts at the base.

Materials & Methods


6. A pulse was given once (2.5kV for 5.70millisecs) and immediately after,

1ml of SOC (room temp.) medium (Annexure II) was added into the

cuvette.

7. The cells were resuspended by inversion and were transferred to a new

microcentrifuged tube and incubated at 37 0C for 1hr at 150rpm.

8. The cells were obtained as a pellet by a flash spin and plated on LB

Annexure II) + Ampicillin medium.

3.9.3 Screening and sequencing of putative

positive clones

Colonies obtained after an overnight growth of the transformed cells on selective

media were gridded on the same media again and incubated at 370C for 16-

18hrs. These gridded colonies were screened for presence of inserts by PCR. The

positive clone’s plasmid was isolated using the Qiagen kits. Ten clones of

P. ciliata and five clones of P. euphratica were sequenced using ABI automated

sequencer. The sequences are deposited in the European Molecular Biology

Laboratory (EMBL) database or Genbank.

3.9.4 Sequence Characterization of Copia-RT region

1. Similarity searches against the Genebank/EMBL database for the sequenced

clones was performed using the BLAST software of the European

Bioinformatics Institute (EBI).

2. Nucleotide sequences were translated in all the six reading frames using the

EMBOSS Transeq translation tool available online at the EBI website

(http://www.ebi.ac.uk/emboss/transeq/).

3. The amino acid sequences were aligned using CLUSTAL W.

4. The molecular phylogenetics analysis were carried out using the MEGA

version 2.1 software (Kumar 1994).

5. The amino acid divergence between the sequences was calculated using the p-

distance.

6. The phylogenetic tree was constructed based on the p-distance matrix.

3.10 PCR Walking for LTR isolation

The retro-element like sequences were obtained from Dr. W. Boerjan and a PCR

walking strategy was employed to obtain the LTR sequences. Based on the

sequence primers were designed to walk from the RnaseH region to the LTR in

the P. deltoides species. The principle for PCR walking is given in Figure 3.2.

The PCR walking (Siebert et al. 1995) methodology is as follows:

Materials & Methods


3.10.1 Construction of PCR walking Libraries

3.10.1.1 Preparation of Adaptors

Adaptor Oligonucleotide 1 (5’-CTAATACGAC TCACTATAGG GCTCGAGCGG

CCGCCCGGGG AGGT-3’) and adapter oligonucleotide 2 (5’-P-ACCTCCCC-NH2-

3’) were mixed to a final equimolar concentration of 50µM and annealed by

incubation in a boiling water-bath for 5 min followed by gradual cooling to allow

formation of the adaptor duplex. This produces an adaptor duplex, which has

one blunt end for ligation and one 3’ amino end to prevent extension.

The adaptors-oligos were mixed to get a final concentration of 50 M.

Oligo I (100 pm/ l) 12.5 l

Oligo NH2 (100 pm/ l) 12.5 l

Total 25.0 l (final conc. is 50 m)

The tube containing the mix was kept in boiling water for 10 min and then cooled

to room temperature.

3.10.1.2 Restriction of genomic DNA

Separate aliquots of 2.5 g to 3 g good quality, high molecular weight genomic

DNA were digest with EcoRV, DraI, PvuII and ScaI which are blunt end

producing enzymes. Excess enzyme was used to ensure complete restriction of

DNA.

Buffers for each enzyme (from Boehringer Mannheim): EcoRV - Buffer B, DraI -

Buffer M, PvuII - Buffer PvuII or M, ScaI - Buffer H.

1. Four separate restriction reactions were sets were constituted for each of

the four restriction enzymes

2. The PCR mix contained following components with the restriction buffer

being as supplied by the manufacturer for each of the enzymes:

DNA 5.0 l (500 ng/ l)

10 X buffer 10.0 l

Enzyme (50 U) 5.0 l (10 U/ l)

Rnase A (10mg/ml) 1.0 l

Water 79.0 l

Total 100.0 l

Spin for few seconds.

2. Overnight incubation at 37oC was carried out.

Materials & Methods


3. Digested DNA (5 l ) was checked on a 1% agarose gel to ensure complete

digestion.

4. The remaining sample was extracted once with phenol : choloroform : IAA

(25:24:1) and once with chloroform : IAA (24:1).

5. The DNA was precipitated by adding 1/10 volume of 3M sodium acetate (pH-

4.8) and two volumes of ethanol (chilled) or incubate at -20oC for 10-15 min.

6. Centrifugation in a microfuge tube for 10 min was carried out followed by

washing of the pellet with 70% ethanol.

7. The pellet was dried and dissolved in 10 l sterile water.

3.10.1.3 Adaptor ligation

To each of the four tubes of digested DNA the adaptor duplex was added and keep

for ligation according to manufacturers instructions.

1. Ligation reaction consisted of :

Digested DNA 10.0 l

10 X ligase buffer 2.0 l (Boehringer)

T4 DNA ligase 1.0 l

Adaptor duplex 2.0 l (Final conc. will be 5 m)

Water 5.0 l

Total 20.0 l

2. Overnight incubation at 16oC

3. Heat inactivated the T4 DNA ligase by heating at 70oC for 10 min.

4. Added 180 l of water to make the final volume to 200 l and stored at -

20oC. (These 4 sets of reactions constitute the genomic library).

3.10.2 PCR amplification of PCR walk-library for the target region The adaptor ligated DNA was used as a template for two nested PCR amplification

reactions using adaptor primers and target-specific primers. The adaptor primers

are shorter in length than the adaptor and are capable of hybridizing to the outer

primer-binding site.

Two sets of amplifications were performed one after another. The target region

primers were based on the RNaseH region and named as PRH-1 and PRH-2.

Primer sequences:

AP1 Sequence: (27-mer) 5' GGA TCC TAA TAC GAC TCA CTA TAG GGC 3'

AP2 Sequence: (18-mer) 5' CTA TAG GGC TCG AGC GGC 3'

PRH1: (17-mer) 5' CGC ACT AAG CAC ATT GA 3'

Materials & Methods


PRH2: (17-mer) 5' CAT CTC AAA CAT GGC AC 3'

3.10.2.1 First PCR amplification

1. The first amplification was by using primer API (adaptor primer) and primer

PRH1 (target-specific primer). For each set, the following were added:

DNA-Adaptor complex 1.0 l

10 X Taq Buffer 0.4 l

High fidelity-Taq Polymerase (3U/ l) 0.4 l

10 mM dNTPs 0.4 l

Primer 1 (API) 0.4 l

Primer 2 (PRH1) 0.4 l

Water 15.4 l

Total 20.0 l

2. One drop of mineral oil was added and PCR amplification was a two step

PCR with the following parameters:

Denaturation 94oC- 1 min

Annealing/Extension 72oC -4 min 7 cycles

Denaturation 94oC - 1 min

Annealing/Extension 65oC - 4 min 32 cycles

3. Ten l was loaded on a 1.5% agarose gel. (Result of the first PCR amplification

was a smear)

4. Ten times dilution of the remaining 10 l of the first PCR reaction product to a

final volume of 100 l was made to be used as template for the second

amplification.

3.10.2.2 Second PCR amplification

1. The 2nd PCR amplification was by using the nested primer APII (adaptor

primer) and the PRH2 primer (target-specific primer). For each set, the

following reaction mix was prepared using Boehringer enzyme:

DNA (Diluted from 1st PCR) 1.0 l

10 X PCR buffer 2.0 l

Taq Polymerase 3U/ l(Expand long) 0.4 l

10 mM dNTPs 0.4 l

Primer 3 (AP2) 0.4 l

Primer 4 (PRH2) 0.4 l

Water 15.4 l

Total 20.0 l

Materials & Methods


2. One drop of mineral oil was added and PCR amplification was by using the

cycling parameters of the 1st PCR.

3. Loaded 5 l on a 1.2% agarose gel.

4. In the second PCR specific fragments were obtained which were different for

the various enzymes.

3.10.3 Cloning and Sequencing of positive clones

1. The specific fragments obtained after second PCR were cloned in pGemT-

easy vector (Promega, USA) and transformed in E. coli JM109 strain by

electroporation technique which has been described earlier.

2. The E. coli colonies were screened for insert by colony PCR amplification

using vector specific primers namely T7 and SP6 primers.

3. The recombinant clones contained inserts of varying sizes depending on the

enzymes used for PCR walking and were visualized on 1.2% agarose gel.

4. The plasmid DNA was isolated for the clones containing the inserts

5. The clones were sequenced using ABI prism 310 genetic analyzer (protocol

in Section 3.7).

3.10.4 Sequence analysis to identify LTR region

1. The sequence homolgy was carried out using the EMBL database. It was

confirmed that the sequences showed homology with the reported Copia-

retro-element like sequences in the databank.

2. Further sequence characterization was carried out to identify the LTR region

in the cloned sequences by translating the sequences using EMBOSS transeq

software of EBI and identifying the conserved RNAseH domain along with

LTR ends.

Table 3.1: List of ISSR primers used in the present study

Primer Sequence

ISSR-UBC 812 5’-GA-GA-GA-GA-GA-GA-GAGA-GA A-3’

ISSR-UBC 814 5’-CT-CT-CT-CT-CT-CT-CT-CT-CT A-3’

ISSR-UBC 818 5’-CA-CA-CA-CA-CA-CA-CA-CA- G-3’

ISSR-UBC 840 5’-GA-GA-GA-GA-GA-GA-GA-GA- YT-3’

ISSR-UBC 842 5’-GA- GA-GA-GA-GA-GA-GA-GA-YG-3’

ISSR-UBC 843 5’-CT CT-CT-CT-CT-CT-CT-CT-CT-RA-3’

ISSR-UBC 848 5’-CA-CA-CA-CA-CA-CA-CA-CA-RG-3’

SAMPL2 (S2) 5’-C-TC-TC-TC-TC-TC-TC-TC-TC-A-3’

Table 3.2. Sequences of selective primers (+1 and +3) employed in

AFLP analysis

Code Sequence

EcoRI+A 5’-GAC TGC GTA CCA ATT C-A-5'

MseI+C 5'-GAC TGC GTA CCA ATT-C-3'

E-AAC 5’-GAC TGC GTA CCA ATT C-AAC-3’

E-AAG 5’-GAC TGC GTA CCA ATT C-AAG-3’

E-ACA 5’-GAC TGC GTA CCA ATT C-ACA-3’

E-ACC 5’-GAC TGC GTA CCA ATT C-ACC-3’

E-ACG 5’-GAC TGC GTA CCA ATT C-ACG-3’

E-ACT 5’-GAC TGC GTA CCA ATT C-ACT-3’

E-AGC 5’-GAC TGC GTA CCA ATT C-AGC-3’

E-AGG 5’-GAC TGC GTA CCA ATT C-AGG-3’

M-CAA 5’-GAC TGC GTA CCA ATT C-CAT-3’

M-CAC 5’-GAC TGC GTA CCA ATT C-CAC-3’

M-CAG 5’-GAC TGC GTA CCA ATT C-CAG-3’

M-CAT 5’-GAC TGC GTA CCA ATT C-CAT-3’

M-CTA 5’-GAC TGC GTA CCA ATT C-CTA-3’

M-CTC 5’-GAC TGC GTA CCA ATT C-CTC-3’

M-CTG 5’-GAC TGC GTA CCA ATT C-CTG-3’

M-CTT 5’-GAC TGC GTA CCA ATT C-CTT-3’

Table 3.2. Sequences of selective primers (+1 and +3) employed

in AFLP analysis

Code Sequence

EcoRI+A 5’-GAC TGC GTA CCA ATT C-A-3'

MseI+C 5'-GAC TGC GTA CCA ATT-C-3'

E-AAC 5’-GAC TGC GTA CCA ATT C-AAC-3’

E-AAG 5’-GAC TGC GTA CCA ATT C-AAG-3’

E-ACA 5’-GAC TGC GTA CCA ATT C-ACA-3’

E-ACC 5’-GAC TGC GTA CCA ATT C-ACC-3’

E-ACG 5’-GAC TGC GTA CCA ATT C-ACG-3’

E-ACT 5’-GAC TGC GTA CCA ATT C-ACT-3’

E-AGC 5’-GAC TGC GTA CCA ATT C-AGC-3’

E-AGG 5’-GAC TGC GTA CCA ATT C-AGG-3’

M-CAA 5’-GAC TGC GTA CCA ATT C-CAT-3’

M-CAC 5’-GAC TGC GTA CCA ATT C-CAC-3’

M-CAG 5’-GAC TGC GTA CCA ATT C-CAG-3’

M-CAT 5’-GAC TGC GTA CCA ATT C-CAT-3’

M-CTA 5’-GAC TGC GTA CCA ATT C-CTA-3’

M-CTC 5’-GAC TGC GTA CCA ATT C-CTC-3’

M-CTG 5’-GAC TGC GTA CCA ATT C-CTG-3’

M-CTT 5’-GAC TGC GTA CCA ATT C-CTT-3’

Table 3.3 Sequences of SAMPL primers

Code Sequence

SAMPL-1 5’-C-TC-TC-TC-TC-GT-GT-GT-GT-G-3'

SAMPL-2 5’-C-TC-TC-TC-TC-AC-AC-AC-AC-A-3'

SAMPL-3 5’-G-TG-TG-TG-TG-AG-AG-AG-AG-A-3'

SAMPL-4 5’-C-AC-AC-AC-AC-AG-AG-AG-AG-A-3'

SAMPL-5 5’-GA-GA-GA-GA-GA-GA-GA-GA-TA-T-3'

SAMPL-6 5’-A-CA-CA-CA-CA-CA-CA-CA-TA-TA-A-3'

SAMPL-7 5’-T-GT-GT-GT-GT-GT-GT-GT-AT-AT-3'

SAMPL-8 5’-CT-CT-CT-CT-CT-CT-CT-CT-AT-A-3'

B B

Gene Gene

A A

PCR amplification B

PCR product

NTS Spacer

NTS Spacer

A

Figure 3.1 The schematic representation of strategy to amplify the Non Transcribed Spacer (NTS) of the tandemly repeated 5S rDNA. The primers are from the conserved regions of the gene and are marked as ‘A’ and ‘B’ in the Figure. The PCR product comprising of the NTS was subsequently cloned and sequenced.

PCR Walk “Libraries”

Amplify target sequence

from all five libraries

Genomic DNA

fragment

N

N

Primary or First PCR

Second or nested PCR

Examine products on

Agarose gel

Clone major PCR products

AP1

PRH-1

AP2

PRH-2

AP1

AP2

PRH-2

PRH-1

Digest aliquots with restriction enzymes

Ligate to Adaptors

Genomic DNA

Figure 3.2 Schematic representation of PCR Walking strategy

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