MOLECULAR CHARACTERIZATION OF SOME NON-CODING REGIONS OF THE GENOME OF A TELEOST FISH

PiSSERTATION

SUBMITTED FN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF

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By

ABUZAR ANSARI

DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2009

DS4022

DLDICATLD TO MY 5LLOVED T A R ^ N T S

D E P A R T M E N T O F Z O O L O G Y ALIGARH MUSLIM UNIVERSITY

A L I G A R H - 2 0 2 002, INDIA

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I certify that the work presented in this dissertation entitled "Molecular

characterization of some non-coding regions of the genome of a teleost

fish" has been carried out by Mr. Abuzar Ansari under my supervision

and it is suitable for the award of the M. Phil. Degree in Zoology of

Aligarh Muslim University, Aligarh.

Prof. Iqpal Parwez Department of Zoology Aligarh Muslim University Aligarh, India

ACICNOWLLDGME.NT5

Jirst qfalCan higfdy indebted and grateful to my Lord 'SMmghiy SMiak' -who bUssed me udtft

tfie innumerabCe favour of academic xvorfi^and produced it in this present form.

I zvouCd Cike to aclqiozvUdge the kind assistance and guidance extended to me by (Prof. Iq6(U (Parwez to compete my research zcorl^ 9{e thoroughly guided me throughout the research

period, never accepting Cess than my best efforts. 9{is critique made me rethink^and reformidate

iarge portion of this work^and shaped the direction of my current research.

I have great passion to record my sincere thanks to 'Brof. ^iSsar Mustafa "Khan, 'Dean and

Chairman, Jaculty of Life Sciences, "Department of Zoology, J^.iM.IJ., Migarh for providing

the necessary facdities during my research luork^

Ihanks are due to "Dr. Olina 'Panvez, 9dr. Usman J^nsari, "Dr. Suneet Singh, Thr, %%

Zlpadhyay, "Dr. (B.'B. 9^am, "Dr. y^armanur l^ehman !Kfian, Dr. Zubair Slhmad, Dr. "Riaz

lAhmad, lAmir'Bhai and 9{Uofer Slapa from whom I received morcdsupport and best iinshes.

I TVouCd like to ackjwzvtedge my indeStness to Mohd. Omaish JAnsari for his help, vcduable

suggestions and encouragement during the entire period of this ivork-

I am grateful to my Cab coCUgues, Dr. Jauzia J^. Shenvani, 9dr. Mustafa J^rif "Kamal,

Miss. Subuhi Slbidi, Mr. Mohammad iCyas, Dr. Deepali PathakiMrs. CS(isha Sharma and Mr.

Jalal Zlddin Slbbas for their good and cordial company, healthy discussions, timely

cooperation, encouragement and helping attitude throughout my research period.

I am grateful to Mrs. 9lgsreen (Seminar Librarian), for providing books during the course of

study. 1 e?qrress thanks from bottom of my heart to all the non teaching staff of this

department for their help.

My acfqioivledgement remains incomplete zi/ithout mentioning the names of my friends. 1 feel

lucky to acknoivledge the names of 9{azrul, Omair, lAzeem, lAmir, Avnish, Santosh,

Unubhazva, 'Kgshif, Tankgj, Isha, Sajid, "Panvez, ylsim Jaraz, ShoaiS, Zeeshan, "Rizivan and

Zulfiqar for providing the boost of morcd support.

finally I would to thanki my parents, my brothers- Hbuzia and Abuzafar, my sisters and

brother-in-lazvs for their love and support without which I certainly wotdd not have

succeeded. They have been there for me emotionally intellectually and financially, without

their persistence, comprehension and encouragement this dissertation could not have been

lAUgcffh, 2009 MuzarJlnsan

L15T or CONTENTS

CONTENTS P. No.

Dedication i Certificate ii Acknowledgment iii List of Content iv List of Abbreviations v List of Figures vi List of Tables vii

Introduction 01-17

Review of literature 18 - 27

Objectives of the study 28 - 29

Materials and Methods 30 - 45

Results 46 - 62 PART- A: Quantitative and Qualitative analysis of DNA

extracted from the male & female teleost fish C batrachus

PART- B: RAPD-PCR analysis of C batrachus using Three different Primers

Discussion 63 - 71

References 72 - 84

Appendices 85 - 86

IV

LI5T o r A55Kn.VlATION5

AFLP: Amplified Fragment Length Polymorphism

bp : Base pair

BSP : Band Sharing Probability

Cb. : Clarias batrachus

DNA : Deoxyribose Nucleic Acid

EDTA: Ethylene Diamine Tetraacetic Acid

Kb : Kilo base

LINEs: Long Interspersed Nuclear Elements

LTRs : Long Terminal Repeats

mM : Mili Molar

mtDNA: Mitochondrial DNA

nDNA: Nuclear DNA

ng : Nano gram

PCR : Polymerase Chain Reaction

PIC : Polymorphic Information Content

pM : Pico moles

RAPD: Random Amplified Length Polymorphism

RFLP: Restriction Fragment Length Polymorphism

RT : Room Temperature

SINEs: Short Interspersed Nuclear Elements

SNP : Single Nucleotide Polymorphism

SSCP : Short Sequence Conformation Polymorphism

SSRs : Short Sequence Repeats

STRs : Short Tandem Repeats

TE : Tris EDTA

VNTR: Variable Number Tandem Repeats

^I : Micro liter

LI5T o r riGURE-5

Fig. no. Figure details

Fig. 1 The per cent component of various functional and non-functional DNA sequences

Fig. 2 Lateral view of Clarias hatrachus

Fig. 3 Ventral veiw of male and female Clarias batrachus during breeding season

Fig. 4 Flow chart of extraction of genomic DNA of the teleost fish C batrachus from blood with High Salt Method

Fig. 5 Flow chart of RAPD-PCR fingerprinting methodology

Fig. 6 Minigel no.l, 2 and 3, resolved in 1 % agrose gel containing ethidium bromide and run at 70 volt for 2.5 hr.

Fig. 7 RAPD profile of Clarias batrachus generated with Standard PCR (aimealing temperature 36°C) using primer OPA-14 with 2.0 mM MgCb

Fig. 8 RAPD profile of C. batrachus generated with temperature gradient PCR (34 - 51°C) using primer OPA09 with 1.5 mM MgCb

Fig. 9 RAPD profile of C. batrachus generated with temperature gradient PCR (34 - 5 rC) using primer OPA14 with 2.0 mM MgCIa

Fig. 10 RAPD profile of C. batrachus generated with temperature gradient PCR (34 - SrC) using primer 0PA16 with 1.5 mM MgCb

Fig.ll RAPD profile of C. batrachus generated with temperature gradient PCR (50 - 6 rC) using primer 0PA16 with 1.5 mM MgCb

Fig. 12 RAPD profile of C batrachus generated vdth temperature gradient PCR (34 - 5 rC) using primer OPA16 with 2.0 mM MgCh

Fig. 13 RAPD profile of C. batrachus generated with temperature gradient PCR (34 - 5 rC) using primer 0PA16 with 2.5 mM MgCb

Fig. 14 Dendrogram showing the plylogenetic relationships among different individuals of C. batrachus

VI

Ll5TOrTABLL5

Table no. Table details

Table 1 Details of the primers used in the present study

Table 2 Standard temperature (36°C) RAPD-PCR template

Table 3 Temperatures gradient (34 - 51°C) RAPD-PCR template

Table 4 Temperatures gradient (50 - 6 rC) RAPD-PCR template

Table 5 DNA Concentration, purity and yield of the 24 specimens of C. batrachus

Table 6 Details of Minigel no. 1,2 and 3

Table 7 Samples detail of RAPD-PCR done at annealing temperature 36°C with primer 0PA14 (corresponding profile is given in Fig. 7)

Table 8 Samples detail of RAPD-PCR done at armealing temperatures range 34-51°C with primer OPA09 (corresponding profile is given in Fig. 8)

Table 9 Samples detail of RAPD-PCR done at annealing temperatures range 34-51°C with primer OPA14 (corresponding profile is given in Fig. 9)

Table 10 Samples detail of RAPD-PCR done at annealing temperatures range 34-51°C with primer OPA16(corresponding profile is given in Fig. 10)

Table 11 Samples detail of RAPD-PCR done at annealing temperatures range 50-61°C with primer OPA16(corresponding profile is given in Fig. 11)

Table 12 Samples detail of RAPD-PCR done at annealing temperatures range 34-51°C with primer OPA16(corresponding profile is given in Fig. 12)

Tablel3 Samples detail of RAPD-PCR done at annealing temperatures range 34-51°C with primer 0PA16 (corresponding profile is given in Fig. 13)

Table 14 Details of different combinations of DNA, primer and MgCli concentration and their response

Table 15 Matching Matrix

Table 16 Matching coefficients values showing the genetic similarity among 12 individual of C. batrachus

VII

INTRODUCTION

INTKODUCTION

The Eukaryotic genome is complex and haploid genome consists of

approximately 3 billion base pairs encoding an estimated 30,000 genes. The

genome may be divided into different classes based very broadly on

functional properties, i.e. fiinctional the 'Coding' and nonfunctional the

'Non-coding' regions (Fowler et al. 1988). The 'Coding' region is called as

exon and the 'non-coding', intra-genic segment is called intron, present

within the coding sequence. Thus, eukaryotic genes often include interrupted

gene structures with multiple exons. Structurally the genome consists of

gene related sequences and extragenic DNA. The gene related sequences

consist of about 30 percent (in human). Only a small portion of the genome

is organized into genes, less than 3 percent of the genome specifies protein

products. Another 27 percent is present as introns, promoters, pseudogenes,

and gene fragments. The vast majority of nucleotides -70 percent compose

extragenic sequences. Two forms of extragenic sequences are predominant:

Unique sequences and Repetitive sequences (Bannett 2000).

1. Unique sequences are the nonrepetitive DNA, i.e. there is only one copy

present in a haploid genome. A very typical example is the gene for

a-globin, which occurs in 1-3 copies in human genome (Orgel and Crick,

1980).

2. Repetitive sequences are present in more than one copy in a haploid

genome, like 300 copies of Alu repeats. These repetitive sequences are

also known as 'junk', 'selfish' or 'parasitic' DNA (Orgel and Crick,

1980). Eukaryotic genome consists of various types of repetitive DNA.

I. Types of Repetitive Sequences

On the basis of number of copies of a specific sequence, repetitive DNA

sequences are classified into two types: Highly Repetitive Sequences and

Moderately Repetitive Sequences.

A. Highly Repetitive Sequences

Highly Repetitive Sequences consist of short sequences, repeated many

times in "tandem repeats" in large cluster. Because of its short repeating

unit, it is also known as simple sequence repeats (SSR). These repeats

consists Satellites, Minisatellites and Microsatellites.

B. Moderately Repetitive Sequences

Moderately Repetitive DNA composed of short "interspersed repeats"

and dispersed at multiple sites throughout the genome. Moderately

repetitive DNA consists of mobile DNA elements (transposable

elements) which has the ability of 'jumping' to different genomic

locations (Brown, 2002). Mobile DNA elements consists Transposons

(DNA transposons) and Reterotransposons (SINEs, LINEs, LTRs).

II. Organization of Repetitive DNA

On the basis of their mode of amplification, two main organization of

repetitive DNA sequences are tandem repeated sequences and interspersed

repeated sequences (Slamovits and Rossi, 2002). Fig. 1 shows brief detail of

different types of repetitive sequences.

A. Tandemly repeated sequences

Tandemly repeats consists repeats arrays of two to several thousand

sequences units arranged in a head to tail fashion. Tandem repeats are

divided into three subgroups according to the length and copy number of

the basic repeat units as well as its genomic localization.

• Satellite DNA

Satellite DNAs are species specific and widely dispersed throughout the

genome. A single genome can contain several different types of satellite

DNA like Sat I, Sat II and Sat III, each with a different repeat unit. Size

of the basic repeat unit is usually 5 to several hundred bp long and

expand from 100 kb to several Mb. Satellite DNAs are located most

often in the heterochromatin associated with the centromeric regions of

chromosomes. They generally do not transcribe. The biological ftmction

is unknown, but it plays some important role in the structural integrity of

the chromosomes especially during the meiotic and mitotic cycles.

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- Minisatellite DNA

Minisatellites are tandemly repeated DNAs which form cluster of

approximately lOObp to 20 kb in length, with 20-50 repeats, each

containing 10-100 bp in length. It can be subdivided in to two types, first

of which is known as Teleomeric, spread on all telomeric regions and

consists of 10-15 kb of hexamer nucleotides mainly TTAGGG (100

copies of the motif in human). It protects chromosomes ends, provides

means of terminal sequence replication and possible role in chromosome

pairing. The second type of minisatellite sequences are Hypervariable

also known as Variable Number of Tandem Repeats (VNTR) spread

throughout genome, but most clustered in teleomeric regions. The basic

repeat unit of hypervariable DNA may vary in length from 6 to >50 bp

and the total expansion size is 100 bp to -20 kb.

' Microsatellite DNA

Microsatellites are also known as Short Tandem Repeats (STRs) or Short

Sequence Repeats (SSRs) distributed almost ubiquitously throughout

genome. Microsatellite clusters are shorter, usually 10 to lOObp, and the

repeat unit usually 13bp or less. The typical microsatellite consists of a 1-

, 2-, 3-, 4-, or 5-bp unit repeated 10 to 20 times.

B. Interspersed repeat sequences

Interspersed repeats are moderately repetitive DNA composed of short

repeats and dispersed at multiple sites throughout the genome.

Interspersed repeats consists various types of mobile DNA elements like

SINEs, LINEs, LTRs, DNA transposons. The most common and most

abundant types of interspersed repeats in vertebrate are: Short

Interspersed Nuclear Elements (SINEs) and Long Interspersed Nuclear

Elements (LINEs).

(i) Short Interspersed Nuclear Elements are 100-400 bp sequences and

not containing any gene. The most common SINEs in humans

frequently contain a site for the restriction enzyme Alul and

consequently are called Alu sequences, are the most abundant

interspersed repeat, consist of a DNA sequence approximately 300 bp

long. Alu repeats are found throughout the primate family.

(ii) Long Interspersed Nuclear Elements have a large range size. LI the

most common repeats belong to LINEs family have a large range size

from 1000 - 7000 bp long. Some 600,000 copies of LI elements occur

in the human genome, accounting for about 15% of total human DNA.

These repeats found in human and most other mammalian species.

These repetitive DNA sequence is an essential component of genomes and

participate in gene regulation through transcription, translation, and

replication (Shapiro and Stemberg, 2005). They are potentially involved in

genome evolution by creating and maintaining genetic variability. Repetitive

sequences are very dynamic component, leads to evolutionary divergence,

which can be used as genetic markers for genome mapping, genotyping,

species diversity identification and phylogenetic inferences and for studying

the process of mutation and selection.

III. Genome Analysis Markers

Markers many molecular markers are available for studying genome

analysis. They are basically classified under two categories: type I markers

are associated with genes of known function, while type II markers are

associated with anonymous genomic segments. Allozyme markers are type I

marker because the protein they encode has known function. Similarly RFLP

markers are type I markers because they were identified during analysis of

known genes. But RAPD markers are type II markers because RAPD bands

are amplified anonymous genomic regions via the polymerase chain

reaction. AFLP markers are also type II markers because they are also

amplified anonymous genomic regions. Microsatellite markers are type II

markers unless they are associated with genes of known function. SNP

markers are also mostly type II markers unless they are developed from

expressed sequences (Liu and Cordes 2004). Basically these are categorized

under two types of markers, protein and DNA, but there are three general

classes of genetic markers are (A) Allozyme, (B) Mitrochondrial DNA and

(C) Nuclear DNA markers, which are frequently used in population genetics

and phylogenetic studies (Okumus and Ciftci 2003).

A. Allozyme Marker:

The allelic variants give rise to protein variants called allozymes that

differs slightly in electrical charge. Allozyme was first described by

Hunter and Markert (1957) who defined them as different variants of the

same enzyme having identical fiinctions and present in the same

individual. It is a classical protein marker for identifying genetic

variation at the level of enzymes, which are directly encoded by DNA.

The general method for detecting allozyme variation includes extraction,

electrophoresis and detection. No marker development phase is

necessary. Allozyme variation provides data on single locus genetic

variation. Differences in the presence/absence and relative frequencies of

alleles as used to quantify genetic variation and distinguish among

genetic units at the levels of population and species. There are many

applications of allozyme markers for individual and stock identification,

systematic, population structure, conservation genetics. However, the

technique has also certain limitations. One major drawback has been the

failure to read genotypes from small quantities of tissue, which makes

allozymes inapplicable for small organisms (e.g. larvae) and there is

important demand relating to the freshness of tissue samples, which

requires sacrificing the individual and cryogenic storage to preserve

enzyme activity are serious constraints.

B. Mitochondrial DNA (mt DNA) marker:

Mitochondrial DNA marker is the first marker in the evolution of DNA

based markers (Avise et al. 1979). Mitochondria DNA genomes are

haploid and maternally inherited. The D-loop region of mtDNA consists

non-coding regions and highly variable than the rest of the mitochondrial

genome and is, therefore, a very useful marker for the study of very

recently divergent population or species (Parker et al. 1998). It is a more

powerful tool than allozyme because of its smaller effective population

size, making it more liable to genomic drift and greater evolutionary rate

(Ward and Grewe 1995). Variation at mtDNA analysed mainly with two

different approaches, i) RPLP analysis of whole purified mtDNA

obtained from fresh tissue by digesting it with restriction endonucleases;

ii) RFLP analysis of small segments of the mtDNA molecule obtained by

means of PCR amplification (Billington 2003). It has become a very

popular marker and dominated genetic studies designed to calculate

phylogeny and population structure and evolutionary studies.

Mitochondrial DNA studies particularly for stock identification and

analysis of mixed species provide critical information for use in the

conservation and management programmes. Mitochondrial DNA markers

have some disadvantages like strict requirement of fresh or frozen tissue,

necessity to sacrifice the £inimals and need more sample and require some

laborious steps of purification of mtDNA. It reflects only the maternal

lineal history and reveals the low level polymorphism in some species.

C. Nuclear DNA (nDNA) markers:

The most recent approaches to gathering data is from the assessments of

nuclear DNA (nDNA) sequence variation with different nuclear DNA

markers like RFLP, AFLP, SSLPs, SNP and RAPD.

• Restriction fragment length polymorphism (RFLP)

Restriction fragment length polymorphism markers were regarded as

the first landmark in the genome revolution. A molecular marker based

on the differential hybridization of cloned DNA to DNA fragments in a

sample of restriction enzyme digested DNA (Botstein et al. 1980)

specific to a single clone/restriction enzyme combination. Restriction

endonucleases are bacterial enzymes that recognize specific 4, 5, 6, or 8

bp nucleotide sequences and cut DNA wherever these sequences are

encountered, so that changes in the DNA sequence due to indels, base

substitutions, or rearrangements involving the restriction sites can result

in the gain, loss, or relocation of a restriction site. Digestion of DNA

with restriction enzymes results in fragments whose number and size

can vary among individuals, populations, and species. Fragments were

separated on agrose gel electrophoresis and digested genomic DNA

transferred to a membrane using Southern blot analysis, and visualized

by hybridization to specific probes. The major strength of RFLP

markers is that they are codominant markers i.e., both alleles in an

individual are observed in the analysis. RFLP applied in genome

mapping, localization of genetic disease genes, genetic fingerprinting,

paternity testing, in characterization of genetic diversity and breeding

patterns in animal populatio ns biology. The major disadvantage of

RFLP is the relatively low level of polymorphism, difficult with long

steps and time-consuming to develop markers. Thus it has almost

completely been replaced with newer techniques.

• Amplified Fragment Length Polymorphism (AFLP)

Amplified Fragment Length Polymorphism generated by a combination

of restriciton digestion and PCR amplification first employes by Vos et

al. in 1995. AFLPs are dominant and, several markers and alleles are

confounded in the same polyacrylamide gel. It combines the strengths of

RFLP and RAPD markers and overcome their problems. The approach is

PCR-based and requires no probe or previous sequence information as

needed by RFLP. The major advantage of AFLPs is that a large number

of polymorphisms can be scored in a single polyacrylamide gel without

the necessity for any prior research and development. Similar to RAPD,

AFLP analysis allows the screening of many more loci within the

genome in a relatively short time and in low-cost way. AFLP has become

widely used for the identification of genetic variation in strains or closely

related species of plants, fiingi, animals, and bacteria. The AFLP

technology has been used in criminal and paternity tests, in population

genetics to determine slight differences within populations and in linkage

studies to generate maps for quantitative trait locus (QTL) analysis. The

methodology is difficult to analyze due to the large number of unrelated

fi-agments that are visible (on the gel) along with the polymorphic

fi-agments (as with RAPD's).

11

• Short Sequence length Polymorphism (SSLPs) or VNTRs

Short Sequence length Polymorphisms are arrays of repeat sequence that

display length variation. Different alleles containing different numbers of

repeat units. These repeated sequences are the most important class of

repetitive DNAs. They repeat in tandem; vEiry in number at different loci

and different individuals dispersed throughout the genome. Based on the

size of the repeat unit two main classes of SSLPs are Minisatellites and

Microsatellites (Fowler et al. 1988). The term VNTR is frequently used

for both Mini- and Microsatellites. These markers have a vital role in

population genetic.

a) Minisatellite DNA composed of short sequences that are repeated in

teandem, scattered all over the genome and is thought to be

noncoding. Minisatellites are two types multilocus and single-locus

(Parker etal. 1998).

i. Multilocus minisatellites loci are highly variable and useful in

parentage analysis or for marking individual families with high

level of polymorphism. Polymorphism for minisatellites loci are

detected by genomic DNA digestion with 4-cutter restriction

enzyme, separating resulting fragments by agrose gel

electrophoresis, Southern blotting to nylon, and hybridizing to

12

repeat sequences probes to identify fragment length differences

that arise from variations in repeat number. Unfortunately the

highly variable loci are less useful for discriminating populations if

large sample sizes are used. Large numbers of alleles can also lead

to difficulties in scoring and interpretation of data.

ii. Singlelocus minisatellite, the ideal marker to study both

individual identification and genetic variability within and among

population are single loci that are highly polymorphic. Analysis

involves library construction, screening, and sequencing for probe

development, followed by sequential gel blot hybridization until

sufficient resolution is achieved. The products are scored after

staming the gel in ethidium bromide. They have proved very

successful in detecting genetics variations within and between

population differences. Recently minisatellites have been applied

for genetic identity, forensics and identification of varieties.

b) Microsatellite DNA: Microsatellite is a simple DNA sequence that is

repeated several times at various points in the organism's DNA. Such

repeats are highly variable enabling that location (polymorphic locus

or loci) to be tagged or used as a marker. Microsatellite loci are

13

analyzed by amplifying the target region using PCR, followed by

electrophoresis through an acrylamide gel to allow resolution of

alleles that may differ in size by as few as two base pairs.

Microsatellite DNA is extremely useful for population studies.

These VNTRs markers also called "simple sequence repeats (SSRs)".

They are among the fastest evolving genetic markers, with

mutations/generation. The much higher variability at microsatellites

results in increased power for a number of applications.

Microsatellites are becoming increasingly popular in forensic

identification of individuals, and determination of parentage and

relatedness, genome mapping, gene flow and effective population size

analysis, phylogenetics, evolutionary relationship, population genetic

structure, conservation of biodiversity.

' Single Nucleotide Polymorphisins (SNP)

Single nucleotide polymorphisms based on detection of mdividual

nucleotide polymorphism caused by pomt mutations (Hecker et al. 1999).

That give rise to different alleles containing alternative bases at a given

nucleotide position within a locus, due to single nucleotide substitutions

(transitions > transversions) or single nucleotide insertions/deletions.

These variants can be detected employing PCR, microchip arrays and

14

fluorescence technology. Major applications of SNPs in fisheries are

genomic studies and diagnostic markers for diseases.

• Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA marker based on the differential

PCR amplification of a sample of DNAs from short oligonucleotide

sequences (Williams et al. 1990, Welsh and McClelland 1990). This

technique is based on the PCR amplification of random segments of

genomic DNA using a single, short oligonucleotide primer of arbitrary

sequence. A small amount of genomic DNA, one or more oligonucleotide

primer (usually about 10 base pair in length), fi-ee nucleotide and

polymerase with a suitable reaction buffer amplified the DNA at a lower

annealing temperature in a thermal cycler. Reaction products are

analyzed by electrophoresis on agrose gel. The polymorphisms detected

in the nucleotide sequence can then be used as genetic markers. As in the

other DNA markers, the RAPD technique has some drawbacks, in this

case the dominance of markers and reproducibility. Nonetheless, it has

several advantages over other molecular markers, such as that it is

simple, rapid and cheap, with high polymorphism, only a small amount

of DNA is required and there is no need for molecular hybridization and

15

most importantly, no prior knowledge of the genetic make-up of the

organism is required and has been widely employed in many studies.

RAPD have wide range of applications and is used to evaluate genetic

diversity within and between strains. The RAPD is technique useful for

population genetics, species and subspecies identification, phylogenetics,

linkage group identification, chromosome and genome mapping, analysis

of interspecific gene flow, hybrid speciation, comparative genome

analysis, breeding and conservation programmes.

Each molecular marker has strengths and weaknesses generally based upon

the equilibrium between repeatability, cost and development time and the

detection of genetic polymorphism. The increasing availability of molecular

markers provides the comparative analysis of species, population/stock and

individual identity. But, there is as significant subjective element in the

choice of marker. The main considerations in the choice of a marker can be

summarized as: availability of samples, organelle and nuclear DNA,

sensitivity, availability of marker, rapid development and screening, multi-

locus or single-locus, relative cost. Following the comparison all above

mentioned markers, RAPD marker has been used to explore the molecular

characterization a teleost fish Clahas batrachus as an animal model. It is an

16

economically important species in aquaculture and fisheries. The choice of

RAPD marker was made due to its simplicity, rapidity, cost effectiveness,

and no need for prior knowledge of the genetic make-up of the organism.

17

KCVILW o r TML LlTLRATUKL

KCVILW or TME. LITLKATUKL

During the last five decades a large number of highly informative molecular

genetic markers have been developed for genetic improvement in fishes.

Initial studies in molecular genetics started with allozymes, which was

developed in the 1960s, it is a protein marker and was extensively applied

for stock identification study in the recent past. Method for restriction

digestion and hybridization got developed in 1970s and DNA amplification

and sequencing during 1980s which led to the development of various

classes of DNA-based markers. In early 1980s the first population genetic

studies based on analysis of mtDNA marker emerged (Avise et al. 1979).

RFLP was the first land mark of nDNA marker in 1980s, which is a

powerfijl tool of population genetics studies. Later, with the introduction of

the PCR a number of different nDNA markers emerged, this method is used

to analyze DNA polymorphisms and the notable approaches included such

as PCR-RAPD, PCR-AFLP, PCR-SSCP and PCR-RFLP markers which are

all based on polymorphisms in the genetic codes of different species

(Rasmussen and Morrissey 2008). These genetic markers have their merits

and demerits and thus their choice is restricted. Out of these markers, RAPD

marker was produced by PCR with minimum efforts. This marker allows

1R

detection of DNA polymorphism and is extensively used in various fields

over the last 15 years due to its simplicity and versatility.

RAPD - a versatile marker

RAPD is highly versatile marker, it is a powerful tool applied in various

fields of genetics and in number of different living beings such as

microorganisms, plants and animals.

In microorganisms: The RAPD is applied in clinical microbiology for the

identification and characterization of bacterial species and strains (Carretto

and Marone 1995). Ronimus et al. (1997) discriminated the Thermophilic

and Mesophilic bacillus species and strains. Bielikova et al. (2002)

characterized and identified the Entomopathogenic and Mycoparasitic Fungi

and evaluated the genetic stability of strains, used as active ingredients of

commercial biopesticides. Salem et al. (2006) evaluated the genetic

similarity and phylogenetic relationship amongst four Bacillus thuringinesis

subspecies.

In plants; Chlamers et al. (1992) detected the genetic variation between and

within population of Gliricidia sepium and G. maculata. Howell et al.

(1993) identified and classified Musca germplasm using nine primers while

Bhat et al. (1995) profiled the Musca DNA with 60 random primers.

19

Polymorphism in seven Somaclones legume Lathyrus sativus was estimated

by Mandal et al. (1996). The genetic homogeneity in two Himalayan

endangered species Meconopsis paniculata and M. simplicifolia, was

detected by Sulaiman and Hasnain (1996) described. Fischer et al. (2000)

investigated genetic variation amongst and within 17 population of

Ranunculus reptans and Rajasekar et al. (2006) analyzed the genetic

diversity in rough bluegrass Poa trivilis L.

In animals: Nagaraja and Nagaraju (1995) generated DNA profiling of

thirteen silkworms, Bombyx mori genotypes. Suazo et al. (1998)

distinguished African and European Honey bees {Apis mellifera L.) and

Kumar et al. (2001) estimated the genetic variation in Indian population of

yellow stem borer (Scipophaga incertulas). Costa et al. (2004) profiled the

taxonomic identification of three common marine amphiods (Gammarus) of

European estuaries. The genetic similarity and diversity in two species of

Butterflies was estimated by Sharma et al. (2006). Kinberling et al. (1996)

estimated the genetic differences within and among seven isolated

population of northern leopard frogs, Rana pipiens from Arizona and

Southern Utah. On the avian genome. Smith et al. (1996) evaluated the

polymorphism and relatedness within and among four chicken breeds and

two turkey population, Okumus and Kaya (2005) characterized the genetic

20

similarity among chicken while El-Gendy et al. (2005) elucidated the genetic

variation within and between duck population and estimated the

phylogenetic relationships. In an interesting study Bailey and Lear (1994)

distinguished the Thoroughbred and African horse breeds. RAPD marker

used by Bhattacharya et al. (2003) to estimate the inbreeding in cattle and

Lee et al. (2007) detected the DNA damage in human lymphoblastoid cells.

RAPD markers were also applied in molecular ecology (Hardys et al. 1992),

in forensic species identification (Lee and Chang 1994), and in genetic

analysis of livestock species identification (Cushwa and Mendrano 1996).

Applications of RAPD markers in fish genetics

RAPD marker offers a rapid and efficient method for generating a new

series of DNA markers are usefiil tools in fishes. It gives information on the

genetic structure of fish species which is useful in several population genetic

parameters:

Species and subspecies identification

The RAPD marker is used as potential fish species and subspecies

identification method. One hundred and sixteen specimens from eight

species of fish species identification were analysed by Partis and Wells

(1996). Bardakci and Skibinski (1994) assayed the polymorphism within and

21

between population of three species of the tilapia genus Oreochromis and

four subspecies of O. niloticus. Takagi and Taniguchi (1995) identified the

polymorphism of three species of Anguilla. Callejas and Orchando (1998)

identified three species of Spanish barbel of Barbcus. Williams et al. (1998)

identified the subspecies of Largemouth Bass {Micropterus salmoides) and

their intergrades. Prioli et al. (2002) evaluated and identified the genetic

relatedness amongst two Teleostei, Astyanax altiparanae populations.

Brahmane et al. (2008) diagnosed species-specific fi-agment pattern for the

molecular identification of the ornamental aquarium fish species Badis badis

and Dario Dario with RAPD markers.

Monitoring levels of Genetic variation:

When two species are almost indistinguishable morphologically, they are

likely to be easily distinguished genetically. Genetic variation between

species and within species i.e. interspecific and intraspecific variations can

easily be analyzed with RAPD markers for species and subspecies

identification.

" Interspecific genetic variations

Degani et al. (2000) estimated the genetic variability between number of

species of Chichildae family. Das et al. (2005) evaluated and compared

the genetic relationship among six Labeo species at the nuclear DNA

22

variation level while Lakra et al. (2007) detected interspecific genetic

variability and genetic relatedness among five Indian sciaenids.

• Intraspecific genetic variations

The intraspecific genetic variation was evaluated by Caccone et al.

(1997) to identified genetic differentiation within the European Sea Bass.

Mamuris et al. (1998) estimated intraspecific genetic variation in red

mullet (Mullus barbatus) and Alam and Khan (2001) in the Japanese

Loach {Misgurnus anguillicaudatus). Wei et al. (2006) evaluated in rice

field Eel {Monopterus albus) for genetic diversity.

Population genetics

The main objectives of population genetic studies are to characterize the

level of genetic variation within species and account for this variation.

Lynch and Milligan (1994) described several population-genetic parameters

(gene and genotype fi-equencies, within and between-population

heterozygosities, degree of inbreeding and population subdivision, and

degree of individual relatedness) along with expressions for their sampling

variances. Yoon and Kim (2001) evaluated the genetic similarity and

diversity of two different populations of cultured Koran catfish Siturus

asotus. Brahamane et al. (2006) delineated the genetic distance among Hilsa

population sampled form six different locations.

23

Inheritance of RAPD

One of the advantages of DNA markers over protein markers is a greater

choice of mode of inheritance. The RAPD markers were shown by the Fl

generation to exhibit dominant Mendelian inheritance and could thus be

used for subsequent genetic linkage mapping. Foo et al. (1995) investigated

the mode of inheritance of RAPD markers in guppy used for subsequent

genetic linkage mapping and inheritance of RAPD marker were studied in

20 inter and intraspecific cross between brown trout and Atlantic salmon

(Elo et al. 1997). Liu et al. (1998) identified inheritance of RAPD in channel

catfish, blue catfish and their Fl, F2 and backcross hybrids and Appleyard

and Mather (2000) investigated the mode of inheritance of RAPD marker in

tilapia Oreochromis mossambicus.

Gene mapping and DNA polymorphism assessing genetic diversity

The genetic linkage map is constructed to identify DNA-based genetic

polymorphism. Liu et al. (1999) identified genetic polymorphism within and

between channel and blue catfish.

Polymorphism in a population is higher revealing higher tolerance power the

environmental conditions and higher genetic diversity. Johnson et al. (1994)

found extensive polymorphism between laboratory strains of Zebrafish.

AUegrucci et al. (1995) detected genetic changes of acclimation of the

24

European sea bass to freshwater reveals DNA polymorphism. Caccone et al.

(1997) identified DNA polymorphism in the European Sea Bass {O. labrax).

Stock discrimination

Smith et al. (1997) discriminated the stock of orange rough, Hoplostethus

atlanticus with allozymes mitrochondrial DNA and random amplified

polymorphic DNA and Gomes et al. (1998) also used the RAPD markers for

stock discrimination of the four-wing flyingfish, Hirundicthys affinis.

Phylogenetic for genetic relationship

Phylogenetic classification exclusively attempts to show relationship based

on reconstruction the evolutionary history of groups or unique genomic

lineages. RAPD has become a standard tool for understanding the

evolutionary history and relationships among species. Mumuris et al. (1999)

estimated the taxonomic relationship between four species of MuUidae

family. Phylogenetic relationship among puffer fish of genus Takifugu was

studied by Lin-sheng et al. (2001) and eight Spanish Barbus species by

Callejas and Orchando (2002).

25

Breeding and Conservation programmes for Stock improvement

Information on genetic variation is essential for breeding and conservation

programmes and for genetic improvement in fisheries not only to improve

effectiveness of fish production but also to maintain genetic diversity.

Wasko et al. (2004) analyzed the level of genetic diversity of fresh water

fish Brycon cephlaus in breeding and conservation programme.

Studies with primers OPA09, OPA14 and OPA16

Various scientists worked with these primers for different aspects on

different fishes. Jayasankar and Dharmalingam (1997) used for the genome

analysis of Sconbroid fishes. Mamuris et al. (1999) used the above markers

for evaluating taxonomic relationships between four species of the MuUidae

family. Yoon and Kim (2001) selected these markers to evaluate genetic

similarity and diversity of two different populations of cultured Korean

catfish Siturus asotus. Islam and Alam (2004) used them to evaluate the

genetic variation of four different population of the Indian major carp Labeo

rothia. Ambak et al. (2006) employed them to focus on the genetic variation

among four populations of Snakehead fish (Channa striata).

26

Molecular characterization of C bartachus

Limited attempts have been made to study the genetic characterization of

C. batrachus using different tools. Daud et al. (1989) evaluated the genetic

variability and relationship of C batrachus with three other Malaysian

catfish using isozymic loci detection. Comparative Karyomorphology of

C. garipinus and C. batrachus has been carried out by Nagpur et al. (2000).

Microsatellite markers have been isolated fi-om C. batrachus (Volckaert et

al. 1999 and Yue et al. 2003). Microsatellite markers have been also isolated

from C. batrachus by Islam and Islam (2007). Huang et al. (2005) used the

RAPD to assess catfish hybridization in C. fuscus, C. mossambicus, and

C. batrachus. In the present study, RAPD markers developed from

C. batrachus have been used to reveal the inter-specific genetic variation

and optimization of maximum amplification conditions varying the

parameters such as annealing temperature and MgCl2 concenfrations which

are the critical determinates of the PCR reaction.

27

05JLCTIVL5 o r TML STUDY

05JE.CT1VE.5 o r THE. STUDY

Fishes are considered as economically important organism in the form of

food and a model for biological research to unravel molecular genetic

variation of evolutionary adaptation. Among the total population of fish in

the world, about U%(2,200 species) are the inhabitants of Indian

subcontinents (Mijkherjee et al. 2002). This percentage gives an idea of

wide range of aquatic biodiversity and thus extremely varied environmental

conditions. Additionally, fisheries and aquaculture provide employment to

millions of the people.

C batrachus is an important fish species found in fi-esh waters of plains

throughout the India and many other countries. It is easy to culture i.e. either

monoculture or polyculture and can tolerate wide range of environmental

conditions. It is consumer's choices due to taste and high protein content. Its

marketability is high and it fetches higher price than carp. Recently, it has

been noticed that the availability of this fish is gradually getting decreased

and the stock shows the high mortality. This may be due to population load,

overfishing, introduction of exotic fishes (mainly due to Afiican walking

catfish, Clarias gariepinus) and other anthropogenic factors. This is

resultmg in the shrinkage of genepool and loss of genetic variability. Hence

28

maintenance of the wild population and management of the cultured stock is

the need of the time. In the view of this, the present study has been planned

to quantify the genetic variability among various individuals of the

C. batrachus to increase its conservation and selective breeding

programmes.

The aim of the present study is to extract DNA from a freshwater teleost

fish, C. batrachus with High Salt Method and to characterize the genome

using RAPD analysis. The brief objectives of the present work include:

PART- A: Quantitative and Qualitative analysis of DNA extracted

from male & female teleost fish, C batrachus

1. DNA extraction with High Salt Method 2. Quantitative estimation with Spectrophotomerty 3. Qualitative assessment with Spectrophotomerty and Mini Gel

Electrophoresis

PART-B: RAPD analysis of, C. batrachus using three different

Primers

1. RAPD analysis of six male and six female fishes following Standard PCR protocol

2. RAPD analysis of the DNA samples following temperature Gradient PCR by enhancing DNA, primer, and MgCb concentration

3. Statistical analysis of Standard RAPD-PCR

With these objectives in view. Part A of this work deals with DNA

extraction with High Salt Method and Part B deals with DNA profilmg of

C. batrachus performed by RAPD, due to its technical versatility.

29

MATLKIAL5 AND MLTHOD5

MATE.KIAL5 AND MLTHOD5

Taxonomic status of the teleost fish Clarias batrachus (Linnaeus, 1758)

Class - Actinopterygii (Ray-fiiuned fishes)

Order - Siluriformes (Catfish)

Family - CXomdiSiQ (Air-breathing catfishes)

Genus - Clarias

Species - batrachus (Asian Walking Catfish)

Morphological features:

There are few varieties of Clarias batrachus (C. batrachus), the normal

coloured which is a slate grey to olive green or either reddish brown to

greyish black. Male are more colourful than the female with dark spot on

the rare of the dorsal fin, the female does not possess this. The size range is

18 - 24 inches and maximum 1190 gm in weight. Body is elongate, broader

at the head and compressed posteriorly. Skin is smooth without scales like in

amphibians. The dorsal and anal fins are long but are separate from caudal.

Pectoral fins with a pungent spine which is rough on its outer edge and

serrated on its inner edge. Upper jaw is a little projected with two pairs of

barbels and lower jaw also contain two pairs of barbels. Occipital process is

more or less triangular (Fig. 2). A dendrictic accessory respiratory organ

30

present dorsal to gills. Genital papilla (Clasper) in males is elongated and

pointed. The bellies of females are prominent than males during spawning

seasons in the month of April to August (Fig. 3).

Characteristic features:

The common name is 'Magur' and also it received a common name the

"Walking Catfish" due its ability to walk overland from pond to pond when

their original habitat dries up. It can live out of water for some time, using its

arborescent breathing organs. It inhabits low land streams, swamps, ponds,

ditches, and rice paddies. It can survive both in fi-eshwater and brackish

water environments (temp, range: 10 - 28°C; pH range: 6.0 - 7.5). It is found

in fresh waters of plains throughout in India in addition to Pakistan, Nepal,

Sri Lanka, Bangladesh and Thailand (Jhinghran, 1983).

It is carnivorous in nature but non-piscivorous and opportunist feeder so it

eats just about anything like insect, larvae, earthworms, shells, shrimps,

aquatic plants and debris. Culture is very easy viz. in small ponds or paddy

fields, either monoculture or polyculture along with silver carp, grass carp,

common carp and three Indian Major Carps. Resilience is high, minimum

population doubling in less than 15 months and vulnerability is low. It is a

palatable and highly nourishing fish food, so it is in great demand and

31

Fig. 2: Lateral view of Clarias batrachus

Fig. 3: Ventral view of male and female Clarias batrachus during breeding season

fetches more price than carp. It possesses high protein (15 %), low fat (1 %)

and high iron content (710 mg/100 g tissue) (Hossain et al. 2006). Due to its

nutritive, invigorating and therapeutic qualities and this is recommended by

physician as diet during convalescence. Marketability is high; it is marketed

live, fresh or frozen.

Collection and care of flshes:

Specimens of C batrachus v^ere collected from the local fish market (Rasal

Ganj) of Aligarh. They were kept in glass aquaria (23" x 10" x 12")

containing fresh tap water (quantity: -15 lit.; average temperature: 27°C;

average pH: 7.1) and the lighting schedule at 12 hr of light (8:00 to 20:00 hr)

alternating with 12 hr of darkness (20:00 to 8:00 hr). Fishes (body weight:

50 - 150 gm; body length: 1 4 - 2 0 cm) were acclimated to laboratory

condition for about 15 days prior to initiation of experiments. During this

period, they were fed daily (at evening) ad Libitum with minced meat.

Water in the aquaria was renewed daily (in the morning) by siphoning off

and replacing simultaneously with fresh tap water adjusted to the laboratory

conditions

32

Blood Collection:

An aliquot of 0.3 to 0.6 ml blood was collected from healthy specimens of

both sexes with the help of a sterile 2 ml syringe containing small quantity

of an anticoagulant viz. 0.5 M EDTA (appendix-A) fitted with 21 gauge

hypodermic needle by puncturing caudal artery and the blood was

immediately transferred in sterile 15 ml falcon tube containing 9.5 ml lysis

buffer (appendix-A) and kept at 4°C for ftirther processing.

Extraction of genomic DNA from Blood

Extraction of genomic DNA was performed with High Salt Method

(Montogmery and Sise 1990) with few modifications (Fig. 4). Collected

blood sample (mixture of blood and lysis buffer) was centrifuged at 3900

rpm for 08 min. at 4.0°C in refrigerated centrifuge (Sigma 2-16K,

Loborzentriftige GmbH, Osterode, Deutschland). Supernatant was discarded

and pellet was suspended in 12 ml 10:0.1 TE (appendix-A), mixed uniformly

on vortex mixer (Personal vortex, Biovortex V-I plus, Biosan, Lativa) and

then 500 ^1 0.5 M EDTA, 20 ^1 Proteinase K (appendix-A) and 500 |LI1 10 %

SDS (appendix-A) was added. It was mixed thoroughly with vortex mixing

and incubated at 50°C in water bath (Universal water thermostat, BWT-U,

Biosan, Lativa) for 4 hr DNA was purified by adding 12 ml of Saturated

Sodium Chloride (appendix-A) after 4 hr incubation. Vortex mixing was

33

done for 30 seconds then centrifiiged at 6000 rpm for 20 min at 4°C.

Aqueous phase was carefully separated in a 50 ml falcon tube. DNA was

precipitated with twice volume of 95 % chilled ethanol (appendix-A). As

soon as ethanol was added white clustered cloudy DNA pellets appeared.

This was then taken out and transferred into an eppendorf tube. The pellets

were washed twice with 70 % ethanol (appendix-A), then air dried and

reconstituted in an appropriate volume of 10:0.1 TE buffer and stored at

4°C.

Note: The recommended storage concentration of stock DNA is 1 mg/ml or

1 g/nl in small aliquots.

• Quantitive estimation with Spectrophotometry

Quality of extracted DNA was estimated by Ultraviolet absorbance

spectrophotometer (Specord 50 PC, Analytical Jena AG, Germany) at

260 nm, at which wave length an absorbance (A260) of 1 corresponds to

50 fig of double stranded DNA per ml (Sambrook Fritsch and Maniatis,

1989). An aliquot 20 ^1 of the DNA solution were diluted in 10:0.1 TE

buffer to final volume of 400 ^1 (20 ^1 stock DNA + 380 fil 10.0.1 TE).

The dilution factor was 20. The following formula was used to determine

the DNA concentration of the reconstituted DNA samples.

Concentration of DNA samples (^ig/ml) = Measured A260 x 50 ng/ml x dilution factor

34

Flow chart of genomic DNA extraction

0.3 - 0.6 ml blood mixed with 9.5 ml lysis buffer

Centrifuged at 3900 rpm for 8 min at 4°C

Separated pellet and resuspended in 12 ml 10:0.1 TE

Vortex mixing for 5 min

Added 500 ^l 0.5 M EDTA, 20 nl Proteinase K and 500 ^1 10 % SDS

Incubated for 4 hr at 50°C

Added 12 ml of Saturated Sodium Chloride Solution

Vortex mixing for 3 sec

Centrifuged at 6000 rpm for 20 min at 4°C

Separated aqueous phase and added twice the volume of 95 % chilled ethanol

White DNA clustered cloudy pellet appeared

Transferred to eppendorf tube and washed twice with 70 % ethanol and kept it for air drying

Reconstituted it with appropriate volume of 10:0.1

Quantitative and Qualitative analysis done with spectrophotometry and minigel run

Stored at 4°C

Fig. 4: Flow chart of extraction of genomic DNA of the teleost fish C. batrachus from blood >\ ith High Salt Method

Qualitative estimation with Spectrophptomerty

The absorbance value ratio of A260:A280, determines the purity of the

DNA, and 1.4 to 1.7 is an acceptable range. If there will any

contamination with protein, the value ratio of A260:A280 will be

significantly less than the values given above.

Qualitative estimation with Minigel Electrophoresis

Quality of DNA was also assessed by 8 well Minigel Electrophoresis

(GENEI, Bangalore). The DNA samples (10 |iil cocktail) was mixed with

1.5 ml of 6 X loading Dye (appendix-A) and electrophoresed on 1 %

agarose gel (appendix-A) containing 3 )il ethidium bromide (appendix-A)

in 1 X TAE Buffer (appendix-A) at 70 volts for 2.5 hr and observed

under the UV light.

35

Random Amplification Polymorphic DNA-Polymerase chain reaction (RAPD-PCR)

Since RAPD is based on the PCR amplification of the polymorphic DNA

using randomly selected primers (single, short and arbitrary sequence

primers upto 10 bases). The following methodology has been adopted

(Fig. 5,).

Selection of Primers

Three randomly selected decamer (10 bases) primers OPA-09, OPA-14 and

OPA-16 (Table 1) were used. All the primers were from Operon

Technologies Inc., U.S.A. (appendix-B). Initially, OPA-14 was selected for

amplification of DNA of 12 individuals of C batrachus (6 males and 6

females) at Standard Temprature (36°C) PCR to examine the intra-species

genetic variations. Finally, three individuals randomly selected fi-om 24

samples of C. batrachus were screened, to get the appropriate annealing

temperature to obtain maximum amplification i.e. maximum number of

bands of DNA, with increased DNA, Primer and MgCl2 concentration at two

different ranges (34 - STC and 50 - 6rC) of Temperature Gradient PCR.

36

Flow chart of RAPD-PCR methodology

DNA (source: Fish blood)

Assessment of DNA Quantity and Quality

Saige

DNA Isolated using Proteinase K digestion High salt extraction & Ethanol precipitation

Store DNA at 4°C

Prepared DNA & PCR Reaction mixture

Loaded sample in PCR machine

Cyck step T«p. CO Ti«w. (MM) IMIIAl 1)1 NArfRATION

l )h \^n R-MION ANNEALING

MNAI KXTENSION

Store PCR products at4°C

Load samples in agrore gel containing ethidium bromide

Agrose gel electrophoresis for 4 hr

Gel analyzed under UV Transilluminator

RAPD-PCR fingerprint

Ml 12 345 6 7 89 10 11 12 Mil RAPD-PCR bands analysed

with gene tool software

Fig. 5: Flow chart of RAPD-PCR fingerprinting methodology

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37

PCR master mixture or reaction mixture

• Standard PCR master mixture contained: A master mixture of

325.5 ^1 containing 32.5 il of 10 X Taq Buffer (appendix-B); 26 il of

2.0 mM MgClj (appendix-B); 6.5 [il (130 pM) of 20 pM primer

OPA14 (appendix-B); 6.5 |ul of 10 mM dNTPs Mix (appendix-B);

4 jul of 5 U Taq DNA polymerase (appendix-B) made in 250 fil of

Milli Q water (Millipore, France). The Standard PCR reaction was

performed in a 23 fil reaction mixture, with the DNA samples of 12

different individuals (6 male and 6 female) of C. batrachus (randomly

selected). Volumes of 1.5 to 2.0 jil (40 - 50 ng) of genomic DNA of

each individual were added in 23 }il PCR master mixture and the total

volume was 25 |itl.

• The Temperature Gradient PCR master mixture contained: A master

mixture of 325.0 il containing 32.5 il of lOx Taq Buffer; 19.5, 26 or

32.5 ^l (1.5 mM, 2.0 mM or 2.5 mM) of MgC12; 12 \i\ (240 pM) of

20 pM primer (OPA09, OPA14 or OPA16); 6.5 |il of 10 mM dNTPs

Mix; 4 )il of 5 U Taq DNA polymerase made in 237 to 250 \i\ of Milli

Q water. The Temperature Gradient PCR reaction was also performed

in a 23 |LI1 reaction mixtures, with the DNA samples of a single

individual of C. batrachus (randomly selected). Volumes of 3 ^1

38

(75 - 95 ng) of genomic DNA of a single individual of C. batrachus

were added in 23 il PCR master mixture and the total volume was

26^1.

Note: In all Temperature Gradient RAPD- PCR only MgCl2 was varied

(1.5, 2.0 or 2.5 mM) while increased DNA concentration (from

45 - 59 ng to 75 - 95 ng) and primer concentration (from 130 - 240

pM) were constant throughout the Temperature Gradient PCR.

Polymerase chain reaction condition

Polymerase chain reaction was performed with three above mentioned

primers. The Standard PCR performed at constant annealing temperature i.e.

36°C (Table 2) of genomic DNA of 12 different individuals of C batrachus,

and the Temperature Gradient PCR performed at two different range of

annealing temperature i.e. 34 - 51°C (Table 3) and 50 - 61°C (Table 4) with

increased DNA, Primer and MgCl2 concentration of genomic DNA of a

single individuals of C. batrachus. A total of 25 )nl PCR reaction mixture

containing genomic DNA of C. batrachus was amplified using thermal

cycler (ATC401, Thermal cycler, Apollo, CLP, U.S.A.).

39

Details of the Standard Temperature and Temperature Gradient RAPD-PCR templates

Table 2: Standard Temperature (36°C) RAPD-PCR template

Stage Cycle Step Temperature (°C) Time (min) Process 1 1 1 94 5 INITIAL DENATURATION

2 45 1 94 I DENATURATION

2 45 2 36 1 ANNEALING 2 45 3 72 2 EXTENSION

3 1 1 72 5 FINAL EXTENSION 3 1 2 4 4 HOLD

Table 3: Temperatures Gradient (34 - SrC) RAPD-PCR template

Stage Cycle Step Temperature (°C) Time (min) Process 1 1 1 94 5 INITIAL DENATURATION

2 45 1 94 I DENATURATION

2 45 2 34-51 1 ANNEALING 2 45

3 72 2 EXTENSION 3 1 1 72 5 FINAL EXTENSION 3 1

2 4 4 HOLD

Table 4: Temperatures Gradient (50 - 6rC) RAPD-PCR template

Stage Cycle Step Temperature (°C) Time (min) Process 1 1 1 94 5 INITIAL DENATURATION

2 45 1 94 1 DENATURATION

2 45 2 50-61 1 ANNEALING 2 45

3 72 2 EXTENSION 3 1 1 72 5 FINAL EXTENSION 3 1

2 4 4 HOLD

40

Agrose gel electrophoresis of RAPD-PCR Products

The amplified products were mixed with 1.5 ml 6 X loading Dye and run on

1.4% agrose gel containing ethidium bromide with two known DNA size

markers in 14 well Midigel electrophoresis (Bio-rad, Hercules, USA). Out of

14 wells, I'' and 14' were loaded with 7 ^1 of ^NA/Hind lU Marker

(appendix-B) and 11 |il of OX174DNA/BsuRI (Haelll) Marker (appendix-

B) respectively. The amplified products of Standard PCR were loaded, fi-om

2 to 7 and 8 to 13 wells with six different male and female respectively of

the same species, while the amplified product of temperature Gradient PCR

were loaded in wells 2 to 13 of the amplified product of the same fish.

Finally, the gel was run at a constant power supply i.e. 70 volt in Ix TAE

buffer for 4 hr.

After 4 hr. of run gel images were captured using Gene Snap Software

(Syngene, England, U.K.) as a permanent record. The bands m the RAPD

profile ranged fi-om higher to lower molecular weight fi-om the origm point

(the wells). The >.DNA/Hind UI and OX174DNA/BsuRI (Haelll) were used

as DNA markers to measure the band size.

The RAPD profiles of temperature gradient gels were analyzed for the

appropriate annealing temperature at which maximum numbers of bands

41

occurred and standard gel was used for estimation of intra-species genetic

variations. Each lane was scored for the presence or absence of every

amplification product and data entered into binary matrix to construct

matching matrix. Matching coefficients of similarity were calculated;

plylogenetic analysis was analyzed using Neighbor Joining Method (Saitou

and Nei, 1987) to construct dendrogram using Gene tool software (Syngene,

England, U.K.).

42

Parameters and Statistical analysis

For statistical analysis, the Standard RAPD-PCR results of the bands detect

by Gene tool software from the gel picture was used to estimate the

following parameters.

(i) Matching coefficients or similarity coefficients obtained by the

comparison between any two Tracks (individuals) on the same gel

image, for the constructing a single data index i.e. genetic similarity

index. The following formula was used for calculating Matching

coefficients (Islam and Alam (2004).

2NAB

Matching coefficient ( N A + N B )

Where: NAB is the total number of bands shared by individuals A and B

NA is the number of bands scored by A

NB is the number of bands scored by B

(ii) 'M' is the mean of the total number of bands obtained through the

gel picture, and scored in binary codes as ' 1' for presence and '0 ' for

absence of a specific amplified band to constructing matching matrix.

'M' was calculated from the following formula:

Total number of bands in the whole gel M=

Total number of individuals (fishes)

43

(iii) *X' is the mean probability that fragment (band) present in one

individual is also present in another. The value of 'X' calculated by

taking the average of the matching coefficients i.e. similarity

coefficients for the similarity index. 'X' was calculated from the

following formula;

Total sum of the matching coefficient values of the half replica

Total number of matching coefficient values of the half replica

(iv) T ' is the probability that the fingerprints of unrelated individuals

are identical, also called Band Sharing Probability 'BSP'. This was

calculated from the following formula:

P or BSP or PIC= (X) ^

Where: 'X' is mean probability

'M' is mean of total bands

(v) 'Q' is the mean allele frequency of bands i.e. the portion of alleles

in a particular population, value was obtained by the following

formula:

X=2Q - Q2

When simplified it gives

Q=l±Vl+X

Where: X is mean probability '

44

(vi) 'H' is the Heterozygosity which signifies as to how much the

individuals are genetically dissimilar from each other. This value was

calculated by the following formula:

H=l-Q

Where: 'Q' is the mean allele frequency

Note: For calculating H, only -ve value of Q was taken

(vii) Cluster Analysis is to determine the degree of genetic relatedness

among different individuals of a species or population. Data for

cluster analysis was obtained by matching coefficient table, which

was obtained by comparing the bands pattern of individuals using the

Neighbor Joining Method.

(viii) Dendrogram constructed by Gene tool software, based on 'Neighbor

Joining Method' (Saitou and Nei, 1987). It is a graphical

representation for displaying tract similarity based on matching

information revealed the genetic distance. They represent hierarchical

relationship using a tree like structure, where the branch lengths

represent the similarity between tracks. The shorter the branch the

more similarity matched is the tracks.

45

KL5aLT5

RE.5ULT5 o r TAKT - A

The result of part 'A' deals with the DNA extraction process which was

done by taking 24 different individual of C. batrachus. The DNA was

extracted from blood of the fishes with High Salt Method consisting of

SDS/Proteinase-K digestion, Sodium Chloride extraction and Ethanol

precipitation. This gave the sufficient amount and good quality of DNA, The

yeild and the quality of DNA of all 24 individuals are as follows:

• Quantity of DNA

The concentrations of DNA obtained from different individuals ranged

from 639.2 to 1316.8 ng/|il (Table 5), which is considered as good yeild.

• Quality and Purity of the extracted DNA

Pure DNA is vital for any molecular biology experiment. The purity of

extracted DNA was checked from the ratio of A260/A280. The ratio of

A260/A280 =1.7 for any DNA is regarded as DNA of high purity. The

ratio at A260:A280 obtained in almost all samples ranged from 1.5 to 1.7

which falls in the acceptable range with minimal levels of other

contanimants (Table 5).

46

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47

• Miligel electrophoresis

The intactness of the DNA of 24 individuals (Table 6, 7 and 8) was

further checked by Minigel electrophoresis. All three Minigels revealed a

single bright blob totally free from any smear, suggesting that the quality

of DNA was good (Fig. 6). This indicates that the extracted DNA is

intact or unsheared. The DNA seems to have migrated completely out of

the loading slot since there was no fluorescence left in the wells which

indicates that the loading and running of gel was proper.

The above quantified values and Minigel electrophoresis clearly show that

High Sah method gave high yield and good quality DNA, which can be

safely used for any PCR run.

48

Table 6: Details of Minigel no. 1,2 and 3

Details of minigel no. 1

Lane no. Fish No. Sex Loaded DNA Cone (ng/fil)

01 Cb-01 M 20.9

02 Cb-02 F 23.0

03 Cb-03 F 12.0

04 Cb-04 M 26.0

05 Cb-05 F 20.7

06 Cb-06 M 22.2

07 Cb-07 F 32.6

08 Cb-08 F 20.1

Details of Minigel no. 2

Lane no. S. No.

Fish No. Sex Loaded DNA Cone. (ng/Ml)

01 Cb-09 M 24.2

02 Cb-10 F 25.4

03 Cb-11 F 25.6

04 Cb-12 M 22.8

05 Cb-13 F 33.7

06 Cb-14 M 33.0

07 Cb-15 F 27.0

08 Cb-16 F 25.0

Details of Minigel no. 3

S. No. Fish No. Sex Loaded DNA Cone. (ng/^l)

01 Cb-17 M 30.0

02 Cb-18 F 29.0

03 Cb-19 F 25.8

04 Cb-20 M 16.3

05 Cb-21 F 28.1

06 Cb-22 M 31.6

07 Cb-23 F 23.3

08 Cb-24 F 29.1

49

Minigel no. 1

Minigel no. 2

Minigei no. 3

Fig. 6: Minigel no. 1, 2 and 3, resolved in 1 % agrose gel containing ethidium bromide and run at 70 volt for 2.5 hr.

KL5ULT5 or PAKT-b

This part deals with the analysis of intra-species genetic variability using

Standard PCR and optimization of annealing temperature using Gradient

PCR with varying MgCl2 concentration for obtaining maximum number of

bands. In this study, three different primers viz. OPA09, 0PA14 and 0PA16

were examined with RAPD-PCR to characterize the genome of

C. batrachus. RAPD-PCR generated varying amplified bands patterns of

low and high intensity produced by the three primers (Fig. 7 to 13) with

different armealing temperatures and of MgCl2 concentration. Standard PCR

was done with OPA14 to estimate the intra-species genetic variability. It is

likely that primer amplification may depend on different annealing

temperatures, with different DNA template, primer concentration and with

varying MgCli concentration. To explore these objectives, three primers

were screened with three different MgCl2 concentrations (1.5, 2.0 and

2.5 mM), and two temperature ranges (34-5 TC and 50-6TC) using gradient

PCR to obtain the optimum amplification to be determined by maximum

number of bands of sharp intensity. The primer and DNA concentration

increased after Standard PCR and was constant throughout the Gradient

PCR. The results of Standard and Gradient PCR are described primer-wise

with varying annealing temperature and MgCl2 concentration.

50

Band pattern of OPA14 of the genomic DNA of six male and six

female catfish C. batrachus generated at Standard annealing

temperature (36"C) with 2.0 mM MgCb.

The gel picture as shown in Fig. 7 generated with primer OPA14 at

constant annealing temperature of 36°C from lane 1-12 with standard

PCR, revealed 2-3 scorable bands (Table 7), in the molecular weight

range of 1124-318 bp. The maximum number of bands i.e. 3 were

observed in both sexes of C batrachus of same molecular weight range

between 1124-318 bp. The most intense band corresponding to 1124 bp

appeared in all 12 individuals and showed the monomorphic band pattern

(constant RAPD bands present in all the individuals in a sample), while

the faintest band corresponding to 318 bp appeared in some individuals

the of lanes 1-7 and disappeared in rest of the individuals. Similarly, one

moderate intensity band of molecular weight of 984 bp appeared in some

individuals of lanes 1-7 and 9-11 and disappeared in the rest individuals.

Bands of 984 bp and 318 bp showed polymorphism on the basis of

presences and absence of band. These two detected polymorphic bands

revealed intra-species genetic variation in C. batrachus.

51

Base pair Base pair

Fig. 7: RAPD profile of C hatrachits generated with Standard PCR (annealing temperature 36°C) using primer OPA-14 with 2.0 mM MgCli, resolved in 1.4% agrose gel containing ethidium bromide and run at 70 volt for 4 hr.

M-I : X DNA/Hind HI, Marker M-D: OXl 74/BusRI (HaelH), Maiker Lanes 1-6: Loaded DNA samples of male Clarias batrachus (Cb.) Lanes 7-12: Loaded DNA samples of female Clarias batrachus (Cb.)

Table 7: Samples detail of ILAPD-PCR done at annealing temperature 36"C (corresponding profile is given in Fig. 7 above)

Lan« No. 1 2 3 4 5 6 7 8 9 10 11 12

Loaded DNA Sample No.

a-04 Cb-06 a-17 a-22 a-23 a-24 Cb-05 Cb-07 a-08 Cb-09 Cb-10 a-16

Max. No. of Bands present

2 2 3 3 3 3 3 2 3 3 2 3

Band pattern of OPA09 generated at gradient annealing temperature

34 - Sl-'C with 1.5 mM MgCh.

The present gel picture (Fig. 8) generated with primer OPA09 clearly

showed the effect of varying annealing temperatures ranging from

34-5 r C with the increment of ~0.6°C from lane 1-12. The gradient

annealing temperatures generated 2 - 4 scoreable bands (Table 8), in the

molecular weight range of 1295 - 479 bp. The maxunum numbers of total

bands observed were 4, which were visible at the annealing temperature

range of 40.1 - 44.9°C in lanes 5-8, in the molecular weight range of

1104 - 479 bp. The most intense band corresponding to 1104 bp appeared

at all temperatures with high intensity, but the intensity virtually declined

at temperatures at 49.9°C and 50.5°C. The faintest band corresponding to

479 bp appeared at temperature range of 40.1 °C - 44.9°C and one band of

molecular weight of 567 bp of little high intensity appeared at the same

temperature range. One moderate intensity band of 579 bp appeared at all

gradient temperature ranges which appeared in all lanes. The intensity

and reproducibility of band of 1104 bp was very high due to high

amplification followed by band of 579 bp. Thus OPA09 responded best

at temperature range of 40.1- 44.9°C.

52

Basepair Base pair

Fig. 8: RAPD profile (34-5r'C) using primer UFAO' gel containing ethidiuni bromi

M-I : A DNA Hind III. Marker M-Il: (I)X174 BusRI (HacUl). Marker Lanes 1- 12: Loaded DNA sample of dVa/va.v/)fl/;-c;t7;as (Cb-09)

Table 8: Samples detail of R.\PD-PCR done at annealing temperatures range 34 -51"C (correspond ing profile is gi\ en in Fig. 8 abov e)

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

Annealing Temp. fC)

34.5 35.1 36.3 38.1 40.1 41.7 43.3 44.9 46.9 48.0 49.9 50.5

Max. No. of Bands present

2 2 3 3 4 4 4 4 2 3 2 3

Band pattern of OPA14 generated at gradient annealing temperature

34 - SVC with 2.0 mM MgClz.

Fig. 9 generated with the changed primer OPA14 and increased MgCl2

(2.0 mM), clearly showed the effect of increased MgCl2 and same

annealing temperatures ranging from 34-51°C with the same increment

i.e. ~0.6°C from lane 1-12. The gradient annealing temperature generated

1-2 scorable bands (Table 9), in the molecular weight range of

1968 - 1047 bp. The maximum numbers of total bands observed were

only 2 which were observed in the annealing temperature of 38.1 °C in

lane 4 and in the molecular weight of 1095-1047 bp. Both bands were

moderately intense and almost disappeared in the remaining gradient

temperature ranges. Band of 1095 bp appeared at temperature range 34.5

- 38.rC in lanes 1 - 4, while band of 1047 bp appeared at 38.1 - 43.3°C

in lanes 4 - 7 . Both bands were amplified only at 38.TC. Thus 0PA14

did not generate sufficient amplification possibility due to the absence

complimentary sequences of the primer.

53

Basepair Basepair

310 2S1 271 234

Fig. 9: RAPD profile of C. batrachus generated witli temperature Gradient PCR (34-5rC) using primer OPA14 with 2.0 mM MgCl2, resolved in 1.4 % agrose gel containing etiiidium bromide and run at 70 volt for 4 hr.

M-I : ^ DNA/Hind m. Marker M-H: OX174/BusRI (Haelll), Marker Lanes 1-12: Loaded DNA sample of Clarias batrachus (Cb-10)

Table 9: Samples detail of RAPD-PCR done at annealing temperatures range 34- 51 °C (corresponding profile is given in Fig. 9 above)

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

Annealing Temp. (°C)

34.5 35.1 36.3 38.1 40.1 41.7 43.3 44.9 46.9 48.0 49.9 50.5

Max. No. of Bands present

1 1 1 2 1 1 1 1 0 0 0 0

Band pattern of OPA16 generated at gradient annealing temperature

34 - SrC with 1.5 mM MgCb.

The present gel picture (Fig. 10) generated with the primer 0PA16

brought out the effect of annealing temperatures, ranging from 34-51°C

with a constant increment of ~0.6°C from lane 1-12. The gradient

annealing temperature yielded only 3 scorable bands (Table 10), in a

molecular weight range of 2647-1562 bp which were clearly observed

only at the annealing temperature of 50.5°C in lane 12. Out of three bands

the 2 bands were moderately intense and corresponded to 2647 bp and

2044 bp, while the faintest band corresponded to 1562 bp. The

amplification of all three bands was very low and generated only at the

maximum temperature i.e. 50.5°C and not at any other lower

temperatures range. The response of 0PA16 at gradient temperature

ranged 34 - 51°C was not satisfactory. Thus the temperature range fiirther

increased to explore the maximum amplification of 0PA16 with same

concentration of MgCl2.

54

Basepair

23130 9416 6557

4361

2322 2027

564

Base pan-

Fig. 10: RAPD profile of C. batrachus generated with temperature Gradient PCR (34-51°C) using primer OPA16 witii 1.5 mM MgCI,, resolved in 1.4 % agrose gel containing ethidium bromide and run at 70 volt for 4 hr.

M-I : X DNA/Hind IH, Marker M-II: OX 174/BusRI (HaelllX Marker Lanes 1-12: Loaded DNA sample oiClarias batrachus (Cb-22)

Table 10: Samples detail of RAPD-PCR done at annealing temperatures range 34- 51 "C (corresponding profile is given in Fig. 10 above)

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

Annealing Temp. (°C)

34.5 35.1 36.3 38.1 40.1 41.7 43.3 44.9 46.9 48.0 49.9 50.5

Max. No. of Bands present

0 0 0 0 0 0 0 0 0 0 0 3

Band pattern of OPA16 generated at gradient annealing temperature

50 - 61°C with 1.5 mM MgCb

The gel picture shown in Fig. 11 was generated by the primer OPA16

with increased annealing temperatures range (50-61°C), clearly showed

no amplification of any single scorable band (Table 11) with the effect of

increased annealing temperatures from lane 1-12 with the increment of

~0.4°C. Because OPA16 responded very badly with increased annealing

temperature range without any amplification, it was proposed to be done

at the increased MgCl2 concentration (2.0 mM) with the previous

gradient annealing temperature range i.e. 34-51°C.

\ ^ c ^ ^ ^ ^

55

Basepair

23130 9416 6557 4361

2322 2027

564

M-I I 2 3 4 5 6 7 8 9 10 11 12 M-II

•

•4

<

Basepair

1353

1078

872

603

310 281 271 234 194

Fig. 11: R.\PD profile of C. batrachus generated with temperature Gradient PCR (50-6rC) using primer OPA16 witli 1.5 mM MgCI,, resolved in 1.4 % agrose gel containing ethidium bromide and run at 70 volt for 4 hr.

M-I rXDNA/Hindin, Marker M-U: OXl 74/BusRl (HaelH), Marker Lanes 1-12: Loaded DNA sanple otClarias batrachus (Cb-22)

Table 11: Samples detail of RAPD-PCR done at annealing temperatures range 50 - 61"C (corresponding profile is given in Fig. 11 above)

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

Annealing Temp. (°C)

50.3 50.7 51.5 52.6 53.9 55.0 56.0 57.1 58.4 59.5 60.3 60.7

Max. No. of Bands present

0 0 0 0 0 0 0 0 0 0 0 0

Band pattern of OPA16 generated at gradient annealing temperature

34 - 51"C with 2.0 mM MgCh.

This gel picture (Fig. 12) generated with the primer OPA16 depicted

showed the effect of increased MgCl2 concentration (2.0 mM) with

varying annealing temperature, ranging from 34-51°C. The gradient

annealing temperature and increased MgCl2 concentration generated

relatively higher number of scorable bands numbering 5-8 (Table 12), in

a molecular weight range of 2591-467 bp. The maximum numbers of

total bands observed clearly were eight which were noticeable in the

annealing temperature at 50.5°C in lane 12, in a molecular weight range

of 2462-276 bp. The most intense band corresponded to 1514 bp, while

the faintest band was 276 bp. The most intensive band of 1514 bp

appeared at all temperature ranges and almost disappeared at 34.5°C. The

intensity increased simultaneously with increased annealing

temperatures. The increased concentration of MgCl2 resulted in enhanced

amplification which is evident from large number of scorable bands as

much as eight recorded in the present experiment. However, when the

concentration of MgCb was fiirther increased from 2.0 to 2.5 mM, there

appeared more amplification though only lower armealing temperature

i.e. 36.3°C.

56

Base pair

23130 9416 6557 4361

2322 2027

Base pair

Fig. 12: RAPD profile of C. batrachus generated with temperature gradient PCR (34-51"C) using primer OPA16 with 2.0 miM MgClz, resolved in 1.4 % agrose gel containing ethidium bromide and run at 70 volt for 4 hr.

M-I : X DNA/Hind m, Marker M-n: OX174musRI (Haelll), Marker Lanes 1-12: Loaded DNA sanple ofClarias batrachus (Cb-22)

Table 12: Samples detail of R,\PD-PCR done at annealing temperatures range 34- 51"C (corresponding profile is given in Fig. 12 above)

Lane No. 1 2 3 4 5 6 7 8 9 10

Annealing Temp. CO

34.5 35.1 36.3 38.1 40.1 41.7 43.3 44.9 46.9 48.0

Max. No. of Bands present

6 7 7 5 6 5 6 6 6 5

Band pattern of OPA16 generated at gradient annealing temperature

34 - SrC with 2.5 mM MgClj.

The present gel picture (Fig. 13) generated with the primer 0PA16

showed the effect of increased MgCl2 concentration (2.5 mM) at gradient

temperature range from 34-51°C with the same increment i.e. ~0.6°C.

The gradient annealing temperature and increased MgCl2 concentration

generated 3-10 scorable bands (Table 13), in a molecular weight range of

2005-395 bp. The maximum number of clearly discernible bands were

10 at the annealing temperature of 36.3°C in lane 3, in the molecular

weight range of 1898-401 bp and the most intense band corresponded to

1514 bp, while the faintest band corresponded to 2462 bp. The most

mtense band of 1514 bp appeared at all temperature range and virtually

disappeared at 49.9 and 50.5°C. The two extra bands were generated at

lower temperature i. e. 36.3°C with increased MgCl2 concentration and

the 8 other bands were almost same molecular weight like in figure 12.

The gel picture also established that the primer generated more

amplification at a particular annealing temperature and sufficient

concentration of MgCl2. Thus 0PA16 had given best amplification at

36.6°C with 2.5 mM MgCl2of C. batrachus DNA.

57

Basepair Basepair

Fig. 13: RAPD profile of C. batrachus generated with temperature Gradient PCR (34-51'C) using primer OPA16 witli 2.5 mM MgCl,, resolved in 1.4 % agrose gel containing etliidium bromide and run at 70 volt for 4 hr.

M-I : X DNA/Hind III, Marker M-II: <I>X174/BusRI (Haelll), Marker Lanes 1- 12: lx>aded DN A sanple of C/anas batrachus (Cb-22)

Table 13: Samples detail of RAPD-PCR done at annealing temperatures range34 -51"C (corresponding profile is given in Fig. 13 above)

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

Annealing Temp.rC)

34.5 35.1 36.3 38.1 40.1 41.7 43.3 44.9 46.9 48.0 49.9 50.5

Max. No. of Bands present

7 9 10 9 8 9 7 9 8 8 4 3

The above analysis generated at different annealing temperature gradient

with different primers and MgCli concentrations showed different

banding patterns. The numbers of scorable bands were 2-4 with OPA-9,

0-2 with OPA16, 0-3 with OPA 16 at 1.5 mM MgClj; 0-2 with OPA14

and 5-8 with OPA16 at 2.0 mM MgCb; and 5-10 with OPA16 at 2.5 mM

MgCl2. The maximum number of bands were 4 with OPA - 9, 2 - 3 with

0PA16 at 1.5 mM MgClj; 2 with OPA14 and 8 with OPA16 at 2.0 mM

MgClj; 10 with OPA16 at 2.5 mM MgClj. (Table 14). The primer

OPA 16 generated with gradient annealing temperatures from 50-6 PC

and with 1.5 mM MgCl2 did not give any single band at any temperature,

while with the same primer i.e. OPA 16 generated at gradient aimealing

temperatures from 34-5 TC with 2.5 mM MgCl2 gave the best resuh with

different banding patterns and yielded scorable bands 3-10 in the

molecular weight range from 2005-395 bp. The maximum number of

band i.e. 10 in the molecular weight range from 2005-395 bp, generated

at temperature 36.3°C. Thus it is evident that to obtain the optimum band

pattern with any specific primer, it is essential that gradient PCR must be

run to establish the most optimum annealing temperature, sufficient

amount of primer, DNA and MgCb concentration.

58

e o a I/)

U U

I 2 (J B O (J

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B a.

z Q C M

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a o

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l is £ 5 -

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s M < OH

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59

statistical analysis of Standard RAPD-PCR

The Standard RAPD fingerprint generated for the 12 different individuals of

C. batrachus using the primer OPA14 is shown in Fig. 7. A total of 28 bands

(Table 15) within the molecular weight range 1124-318 bp were scored. 12

bands were monomorphic, i.e. conserved among the 12 fishes and rests of

the 16 bands were polymorphic, i.e. present in some individuals and absent

in others. The matching matrix cleared the above lines.

The matching coefficient or genetic similarity values (Table 16) obtained

from the mentioned formule described previously on the basis of presence

and absence of bands and the mean of the total number of bands was 2.33.

The bands sharing probability 'P' was 0.37 and its mean was 0.66. The mean

of allele frequency of bands 'Q' was 1.24 (+ve value) and 0.08 (-ve value).

The -ve value (0.08) was used for calculating the Heterozygosity 'H' with

mentioned formula and the value was 92 %, its clear that the genetic

dissimilarity was found to be 92%. The high level of intra-species

heterozygosity i.e. 92% revealed the high polymorphism within species of

C. batrachus.

60

Table 15: Matching Matrix

Pea k

Trac k 1

Trac k 2

Trac k 3

Trac k 4

Trac k 5

Trac k 6

Trac k 7

Trac k 8

Trac k 9

Trac k l O

Trac k11

Trac k12

1 X X X X X X X X X X X X

2 X X X X X X X X X

3 X X X X X X X

Table 16: Matching coefficients values showing the genetic similarity among 12 individual of C batrachus

Track 1

Track 2

Track 3

Track 4

Track 5

Track 6

Track 7

Track 8

Track 9

Track 10

Track 11

Track 12

Track 1 1.000 0.800 1.000 1.000 1.000 1.000 1.000 0.400 0.667 0.667 0.667 0.400 Track 2 0.800 1.000 0.800 0.800 0.800 0.800 0.800 0.500 0.400 0.400 0.400 0.500 Track 3 1.000 0.800 1.000 1.000 1.000 1.000 1.000 0.400 0.667 0.667 0.667 0.400 Track 4 1.000 0.800 1.000 1.000 1.000 1.000 1.000 0.400 0.667 0.667 0.667 0.400 Track 5 1.000 0.800 1.000 1.000 1.000 1.000 1.000 0.400 0.667 0.667 0.667 0.400 Track 6 1.000 0.800 1.000 1.000 1.000 1.000 1.000 0.400 0.667 0.667 0.667 0.400 Track 7 1.000 0.800 1.000 1.000 1.000 1.000 1.000 0.400 0.667 0.667 0.667 0.400 Track 8 0.400 0.500 0.400 0.400 0.400 0.400 0.400 1.000 0.400 0.400 0.400 0.500 Track 9 0.667 0.400 0.667 0.667 0.667 0.667 0.667 0.400 1.000 0.667 0.667 0.400 Track 10 0.667 0.400 0.667 0.667 0.667 0.667 0.667 0.400 0.667 1.000 0.667 0.400 Track 11 0.667 0.400 0.667 0.667 0.667 0.667 0.667 0.400 0.667 0.667 1.000 0.400 Track 12 0.400 0.500 0.400 0.400 0.400 0.400 1 0.400 0.500 0.400 0.400 0.400 1.000

61

Dendrogram constructed by Gene tool software, based on 'Neighbour

Joining Method' (Fig. 14) on the basis of genetic similarity values

ranging from 0.400-1.000 (Table 16) showed the phylogenetic

relationship among 12 individuals of C batrachus. In the dendrogram the

blue hexagon values are the genetic distance which joined with black

lines between most similar individuals. The shorter the branch the more

similar is matching between the individuals. Hence the genetic similarity

of individuals 7 and 6; 4 and 5 were 1 and there genetic distance were

0.000 (in blue hexagon), thus they make a cluster. The genetic similarity

of individual 3 was also 1 and that made the cluster with mdividual 7,6;

4,5 and its genetic distance is also 0.000. Like that the other two close

genetic similar individuals form cluster together.

62

tdiS

- ^ Ul

K»> '<^^

Ufl

{«)•

w >-0

Wll

• O w

1adi2

m U3

M2 H

lffl>^

(UDi I U S

mill J u «

UD (. ^ U 7

QODC' U I

Fig. 14: Dendrogram showing the plylogenetic relationships among different individuals of C. batrachus

Dl5Ca5510N

D15CU55ION

The utmost requirement for any study at the molecular biology level is the

good quality DNA based on high purity, intactness and reasonable yield and

the ease of technique for DNA extraction. There are various sources and

methods available for DNA extraction. Sources of DNA can be-tissues like

hair, blood or semen etc. Extraction of DNA from blood is comparatively

very easy. One of earliest and the various hitherto most widely employed

method of DNA extraction is Phenol-Chloroform method, which ultimately

resulted in the development of commercial Kits marketed by several biotech

companies such as Fermantas. Lately, some workers used High Salt method

and CTAB method (for plants) in many laboratories which has overcome

the disadvantages of Phenol-Chloroform by being less arduous and avoiding

the corrosive use of Phenol. However, there were lurking doubts as to

whether the DNA extracted by High Sah method can be successfully used

for PCR amplification.

However, the present study has clearly established that the DNA extracted

for High Salt method is good enough to be used for PCR amplification. On

the yield parameter, the method used in the present study has turned out to

be the best since it has given the highest yield compared to conventional

63

Phenol-Chloroform and the kit method and the latter giving the lowest yield.

The strength of the Kit method is, however, that it can be used with very

small sample volume and the quality is also reasonably good although the

cost factor, as on today, remains a deterrent.

Among the different tissues used for DNA extraction, blood seems to be the

most appropriate particularly in the case of fish where RBC, imlike in

mammals, are nucleated and hence the DNA yield from this tissue is

enormous. Moreover, the other tissues such as liver, muscles, etc require the

use of extensive detergent to get rid of extra tissues which often result in

shearing of DNA molecules. This study has also shown that the proportion

of blood volume used for DNA extraction vis-a-vis the quantity of the

reagents used are of critical importance since large blood volume with

insufficient or disproportionate reagent quantity may result in the incomplete

digestion and insufficient separation of protein which will ultimately provide

poor quality. If more yield is required, it is suggested that the sample should

be processed in small batches with appropriate blood volume and storing the

rest of the blood in EDTA smeared vial at 4°C which could be conveniently

stored for more than one week without any degradation.

64

Yoon and Kim (2001) also used the High Salt method for extracting the

DNA from the blood of cultured Korean catfish and found high yield and

good quantity of DNA and used it for RAPD study. Thus High Salt method

provides sufficient yield of total DNA and is comparatively simpler, faster,

less laborious and Jess expensive method for DNA extraction.

Conventionally, for the Minigel electrophoresis, the recommended gel

concentration is 0.5-0.2%, ethidium bromide 0.5 ^g/ml, the loaded volume

of DNA samples is 10-100 ng and the run for 30-60 min at high voltage until

the bromophenol blue and xulene cyanol migrate the appropriate distance

(Sambrook et al. 1989). However we performed Minigel electrophoresis

with the gel concentration of 1 %, containing ethidium bromide (0.3 \ig/m\)

and the loaded volume (20-30 ng/|al) of DNA samples at 70 volt for 2.5 hr.

Thus with this amount of sample loading the run was found to be the best,

which is clear from the Minigel pictures which are without any smeary

pattern.

The RAPD analysis is generally used for genome mapping, population

studies, stock identification and is extensively used in genetic variability

studies of inter-species and intra-species. In the present study, the intra-

species genetic variability was assessed on the basis of the presence or

fis

absence of RAPD markers of different molecular weight, which indicated

high genetic variability within 12 individual of C. batrachus which is also

established by the low intra-species similarity (0.4-1). Three bands were

found in which one was monomorphic and two were polymorphic (Fig. 6).

The number of bands were few but the dendrogram (Fig. 14) clearly showed

low inter-specific similarity and high-level of genetic variation within the

individuals. The high genetic heterogeneity (92 %) was, therefore, clearly

indicative of high genetic polymorphism within the individuals of

C. batrachus which should exhibit better tolerance in the environmental

conditions.

The RAPD technique, while being extensively informative about genetic

structure, needs to be practiced with extreme caution. It is heavily dependent

on the factors such as, quality of template DNA, component of amplification

reaction mixture, amplification conditions, primer sequence which influence

the quality and size of the RAPD products generated.

It has been well documented that low concentration of DNA is required for

the optimal PCR because both the primer and dNTPs should be in excess;

and over abundance of template DNA will anneal with template sequence,

rather than their annealing to the primer pair and will also increase the

66

chances of forming non-specific bands. Typically, a single copy gene can be

amplified sufficiently in 30 cycles for less than 0.2 ^g of DNA. Jayasankar

and Dharmalingam (1997) optimized the primer concentration (0.32, 0.64,

1.28 and 2.6 i M) and analyzed 200 ng of DNA and thus from the results he

obtained it was concluded that 0.64 iM is best concentration of primer,

while Yoon and Kim (2001) optimized with 10 ng of template DNA. In our

study the primer concentration was 17pM and DNA template concentration

was 75-95 ng, these concentrations gave good results with primer 0PA16.

Thus from these observations, it can be concluded that low concentration of

DNA is required for optimal PCR.

The other critical variable in the PCR condition is the appropriate choice of

annealing temperature used in PCR which depends on the composition of

primer (G+C% should be 50-60%), the annealing temperature should be

between 1-5*'C lower than its corresponding Tm value. Too low annealing

temperature results in non-specific annealing and, therefore, non-specific

amplification, while too high annealing temperature leads to a reduced yield.

In the present study, three primers viz. OPA09, 0PA14 and 0PA16 with

varying annealing temperature ranging from 34-5 TC were used which

yielded 2-4, 0-2 and 3-10 number of scorable bands respectively. These

bands were confined within the base pairs ranging from 0.39-2.64 kb. This

^7

study clearly brought out the effect of varying annealing temperatures on the

amplification efficiency of PCR reaction. Interestingly, when the annealing

temperature in the case of primer 0PA16 was increased to 50-6rC, there

was no scorable PCR bands while with the same primer at the temperature

range of 30-5 l^c with 2.5 mM concentration of MgC^, it yielded a

maximum of 10 bands at 36.3°C within the base pairs ranging from

0.39 - 2.00 kb. These results clearly show the significance of working out an

optimum annealing temperature which may yield the best PCR

amplification.

Several workers have also used the above primers (OPA09, OPA14 and

0PA16) but at a fixed annealing temperature. Jayasankar and Dharmalingam

(1997) used OPA09, OPA14 and OPA16 primers at the annealing

temperature of 36*'C at the MgCb concentration of 2.4 mM and observed the

occurrence of 0-3 bands. Similarly, Mamuris et al. (1998) used all three

primers at 2.5 mM MgCl2 concentration and recoded 28 bands with OPA09

at 38°C annealing temperature the other two primers failed to yielded

satisfactory amplification. Mamuris et al. (1999) used OPA09 at 5 mM

MgCl2 concentration and recorded 7 bands at 56°C. In another study Yoon

and Kim (2001) used two primers OPA09 and OPA14 similar to the present

study at the annealing temperature 36°C and obtained 10-17 and 6-16 bands

68

respectively. In a RAPD study, on Indian major carp Labio rohita, Islam and

Alam (2004) used OPA09 at the annealing temperature of 34°C and obtained

4-9 bands in the range of 0.28- 1.94 kb. This study did not specify the MgCl2

concentration although they used 50 ng of genomic DNA as the template.

Ambak et al. (2006) focused on the genetic relationship among four

population of Channa striata distributed in Peninsular Malaysia. Among the

other primers, he also used OPA09, OPA14 and OPA16 in the above study.

Curiously, while OPA09 produced successful amplification yielding 1-12

bands in a range of 0.25-1.5 kb at the armealing temperature of 36°C and the

other two primers failed to show any amplification. None of the above

investigators used the gradient PCR to determine the optimum annealing

temperature as has been done in the present study. It is, therefore, difficult to

established the effect of varying annealing temperature on the PCR

amplification efficiency. The variation in the number of scorable bands

observed by the different workers may well be due to the difference in the

species investigated in each study. However, it must be emphasized that the

determination of optimum annealing temperature should be a prerequisite for

any successful RAPD-PCR study.

Magnesium Chloride (MgCl2) is essential as a co-factor of DNA polymerase

in the assay buffer. Higher concentration promotes the amplification of

69

unspecific fragments and also increases the melting temperature, while low

concentration reduces annealing efficiency and the synthesis rate. Thus

insufficient MgCl2 concentration leads to low yields and excess MgCl2

resuhs in the accumulation of non-specific products. Varying the

concentration of MgCb (1.5 - 6.0 mM) in the PCR mixture resulted in both

quantitative differences in RAPD-PCR derived DNA bands and qualitative

changes in the DNA band patterns.

In the present study, the effect of three different MgCl2 concentrations (1.5,

2.0 and 2.5 mM) were examined with OPA16 primer and the best results

were obtained at the concentration of 2.5 mM MgCl2 as it generated the

maximum 3-10 scorable bands. The number of bands reduced considerably

when MgCl2 concentration decreased from 2.0 to 1.5 mM. Despite the fact

that varying MgCl2 concentration plays a critical role in the optimization of

PCR amplification, it is indeed surprising that most of the workers have not

optimized the MgCl2 concenfration and have carried out their studies at an

empirical concentration. The significance of optimizing MgCb concentration

for an efficient RAPD-PCR is fiirther established by the studies of

Jayasankar and Dharmalingam (1997) and Ambak et al. (2006). The former

worked with the MgCl2 concentration of range 2.0-2.6 mM and mM) and got

70

the best result at 2,4 mM concentration whereas the latter explored the range

between 1,5- 5,0 mM and at 1.5 mM MgCl2 concentration as the best.

The salient highlights of the present study are as follows:

1. High Salt method is eminently suited for DNA extraction because of

its ease, simplicity, short duration coupled with high purity and good

yield.

2. The whole blood is the best tissue for DNA extraction particularly m

fish, since it renders clean yield with high purity and is easily

obtained,

3. Optimizing RAPD-PCR conditions is an essential prerequisite for an

efficient RAPD-PCR amplification.

4. It is imperative that an optimum annealing temperature must be

worked out for each primer and species by using gradient thermal

PCR.

5. Optimum MgCb concentration is of paramount importance, since it

substantially affects the amplification as has been clearly established

in the present study and also the studies carried out by few other

workers.

71

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84

ArrLNDICL5

Arrn.ND!X-A

0.5 M EDTA (100 ml, pH 8) 18.61 gm Ethylene Diamine Tetraacetic Acid (Sigma) dissolved in 70 ml of Milli Q water (Millipore), pH was adjusted to 8 with 2N Sodium Hydroxide (Qualigens), made uptolOO ml with Milli Q water. Filtered, autoclaved and store at RT.

1 M Tris (100 ml, pH 8.0) 12.1 Igm Tris base (Sigma) dissolved in 70 ml Milli Q water, pH was adjusted to 8 with Concentrated Hydrochloric Acid (Qualigens), made up the solution to 100 ml with Milli Q water. Filtered, autoclaved and store at RT.

10: 0.1 I E (100 ml) 1 ml Tris from IM stock, 20.0nl EDTA form the 0.5 M stock, made up the solution to 100 ml with Milli Q water. Filtered, autoclaved and store at RT.

Lysis Buffer (250.0 ml) 27.38 gm Sucrose (Merck), 0.25 gm Magnesium Chloride (Qualigens), 2.5 ml of 1 M Tris, 2.5 ml of Triton X 100, made up the solution to 250 ml with Milli Q water. Filtered, autoclaved and store at 4°C.

Proteinase K (1 ml) 20 gm Proteinase K (Sigma), made up the solution to 1.0 ml with Milli Q. Store at -20°C.

10 % SDS (100 ml) 10 gm Sodium Dodecyl Sulphate (Sigma), made up the solution to 100 ml with Milli Q water. Filtered, autoclaved and store at RT.

6 M NaCl (100 ml) 35.64 gm Sodium Chloride (Merck), made up the solution to 100 ml with Milli Q water. Filtered, autoclaved and store at RT.

95 % Chilled Ethanol (100 ml) 95 ml Ethanol (Changshu yangyuan Chemicals), made up the solution to 100 ml with Milli Q water. Store at -20.0°C.

70 % Ethanol (100 ml) 70 ml Ethanol, made up the solution to 100.0 ml with Milli Q water. Store at RT.

6X Loading Dye solution (Fermentas) Store at 4°C for short time and at -20°C for long period (ready-to-use).

Ethidium Bromide (1 ml) 10.0 mg Ethidium Bromide (Sigma), made up the solution to 1.0 ml with Milli Q water. Store at 4°C.

85

Arr^NDix-b

Primer OPA09 (Operon Tech. Inc., U.S.A.) Concentration: 20 pM/\i\ Base sequence: GGG,TAA,CGC,C Storage condition: -20°C

Primer OPA14 (Operon Tech. Inc., U.S.A.) Concentration: 20 pM/|al Base sequence: TCT,GTG,CTG,G Storage condition: -20°C

Primer OPA16 (Operon Tech. Inc., U.S.A.) Concentration: 20 pM/|xl Base sequence: AGC,CAG,CGA,A Storage condition: -20°C

lOXTaq Buffer with KCI (Fermentas) Composition: 100 mM Tris-HCl (pH 8.8 at 25°C), 500.0 mM KCI 0.8% Nonidet P40 Storage condition: -20°C

MgCh (Fermentas) Concentration: 25 mM solution Storage condition: -20°C

dNTPs Mix (Fermentas) Concentration: 10 mM of each Storage condition: -20°C

Taq DNA polymerase (Fermentas) Concentration: 5 u/|il Storage condition: -20°C

Lambda DNA/Hindll Marker (Fermentas) Concentration: 0.1 ^g/^l Molecular weight range: 23130- 125 bp, generated 08 bands (ready-to-use) Storage condition: 4°C

OX174 DNA/BsuRI (Haelll) Marker (Fermentas) Concentration: 0.5 ng/|il Molecular weight range: 1353 - 72 bp, generated 10 bands (ready-to-use) Storage condition: 4°C

86