MITOCHONDRIAL DNA VARIATION OF CULTURED AND WILD

POPULATIONS OF ASIAN SEABASS (Lates Calcarifer) IN THAILAND

THESIS PROPOSAL

YUSMANSYAH

A THESIS PROPOSAL SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE MASTER DEGREE OF SCIENCE IN AQUATIC
SCIENCE
GRADUATE SCHOOL
BURAPHA UNIVERSITY
SEPTEMBER 2008
COPYRIGHT OF BURAPHA UNIVERSITY
 THESIS PROPOSAL

Name : Yusmansyah Examining Committee

Program : Master of Science Wansuk Senanan, Ph.D. (Principal Committee)
Department : Aquatic Science Chuta Boonphakdee, Ph.D. (Committee)
Academic year : 2008 Thadsin Panithanarak, Ph.D. (Committee)

Title : Mitochondrial DNA Variation of Cultured and Wild Populations of Asian

Seabass (Lates calcarifer) in Thailand
 CONTENTS

CONTENTS
LIST OF TABLES
LIST OF FIGURES
CHAPTER
1 INTRODUCTION
1.1.Background …………………………………………………… 1
1.2. Objectives………………………………………………….. 3
1.3. Cotribution to Knowledge ………………………………… 3
1.4. Scope of Study……………………………………………... 3
1.5. Hypothesis.………………………………………………… 4
2 LITERATURE REVIEWS
2.1. Biology of Asian seabass...…………………………………. 7
2.2. Ecology and Distribution...………………………………… 9
2.3. Genetic Processes in Population…………………………… 10
2.4. Mitochondrial DNA as Genetic Marker for Population genetic-
Analysis…………………………………………………… 13
2.5. Molecular Techniques for mtDNA Analysis………………. 15
2.5.1. Restriction Fragment Length Polymorphism…………... 15
2.5.2. Direct Sequencing……………………………………… 16
2.6. Genetic diversity of Asian seabass and fish species with similar life
Histories……………………………………………………. 18
2.7. Genetic data analysis……………………………………….. 23
2.7.1. Genetic diversity within populations…………………... 23
2.7.2. Genetic differentiation among Populations……………. 24
3 RESEARCH METHODOLOGY
3.1. Samples Collection ………………………………………... 29
3.2. DNA Extraction……………………………………………. 30
3.3. PCR Amplification………………………………………… 31
3.4. Enzyme Digestion………………………………………….. 31
3.5. Data Analysis………………………………………………. 33
REFERENCES 35
 LIST OF TABLES

Table Page
2.1 Genetic diversity of the mtDNA in L. calcarifer and species with
similar life histories 21
2.2 General design for hierarchical analysis of molecular variance
(AMOVA) 25
3.1 Population sources and number of samples 30
3.2 Recognition size sensitivity of restriction enzymes used 32
 LIST OF FIGURES

Figure Page
1.1 Linear regression plot of Tamura-Nei’s and delta mu squared
genetic distance with coastal distance inferred from mtDNA
sequencing and microsatellite using Mantel test 5
2.1 Morphology of Adult Seabass/barramundi (Lates calcarifer Bloch) 7
2.2 World geographic distribution of Asian seabass 10
2.3 Outcome of three modes of selection: (A) Directional selection,
(B) Stabilizing selection, and (C) Disruptive selection 13
3.1 A map of sampling locations 29
 1

CHAPTER I
INTRODUCTION

1.1. Background
Asian seabass (Lates calcarifer Bloch), commonly called ‘giant sea perch’
or barramundi, has become economically important coastal, estuaries and marine fish
species in the world, especially in the Indo-pacific region. In 2006, Food and
Agriculture Organization (FAO) estimated the world aquaculture production of L.
calcarifer to reach 31,909 tons with market value US$ 88,383,000. World demand for
L. calcarifer is increasing overtime, not only for flesh but also for fingerlings. It
stimulates rapid growth in aquaculture, particularly cage culture, of L. calcarifer in
Asian countries, mainly Japan, Taiwan, China, and almost all of South East Asian
countries (De Silva & Phillips, 2007). Aquaculture of L. calcarifer in Thailand, pond
or cage culture as well as larval rearing, is also growing and spreading in almost all
provinces along the Gulf of Thailand and Andaman Sea coastlines, (Department of
Fisheries of Thailand, 2005).
In Thailand, L. calcarifer fingerlings are produced mainly in hatcheries. The
fingerling are not only distributed for local fish farms but also exported to several
countries (Interviews with local hatcheries). Well managed breeding program plays an
important role in producing good quality fingerling to meet industrial scale
aquaculture needs. A successful breeding program depends greatly on broodstock
management that maintains high level of genetic diversity of a hatchery population.
Reduced genetic variation within population may lead to undesirable
consequences on traits related to production (inbreeding depression), for example
resistance to diseases stresses, and adaptive potential to environmental stresses
(Frankham, 2005). Loss of genetic variation within-population in hatchery
populations may be a result of inappropriate hatchery practices, such as mating a
limited number of broodstock, and mass spawning causes unequal sex ratio and
unequal contribution of each family (Frost, Evans and Jerry, 2006).
Wild populations can usually serve as potential source of genetic variation
for hatchery populations as they tend to contain higher genetic diversity than hatchery
 2

populations. However, information regarding genetic diversity of hatchery and wild

populations of L. calcarifer in Thailand is remaining unknown. Genetic diversity data
will very useful for designing broodstock management strategies that maintain genetic
diversity, such as broodstock collection, mating schemes, and rearing practices (Le
Vay et al., 2006).
A study in genetic of L. calcarifer in Southeast Asia using microsatellite
marker shows relatively high genetic diversity within both hatchery and wild
populations, but wild populations tended to have higher within-population genetic
diversity than hatchery populations (Zhu et al., 2006). Lower genetic diversity in
hatchery populations is commonly observed in other species, for example Japanese
flounders Paralichthys olivaceus in Japan (Sekino, Hara and Taniguchi, 2002),
Japanese scallops Patinopecten yessoensis in China (Li, Xu and Yu, 2007), Pacific
oysters Crassotrea gigas in Australia (English, Maguire and Ward, 2000) and brown
trout Salmo trutta (Hansen et al., 1997).
Mitochondrial DNA (mtDNA) markers have been useful in describing
genetic variation in hatchery and wild populations, and population structure of several
species such as Giant Tiger Shrimp Penaeus monodon (Klinbunga et al., 2001),
mahseer species (Nguyen et al., 2006b), Common Carp (Thai, Pham, & Austin,
2006), Asian Moon Scallop, Amusium pleuronectes (Mahidol et al., 2007). A mtDNA
is a single ‘chromosome’, consisting 15-60 kilo base pair length (Burger et al., 2003).
Characteristics of mtDNA, such as uniparental inheritance (Birky, Fuerst, &
Maruyama, 1989), smaller effective population size than nuclear DNA (Avise, 2004),
and higher mutation rates (Chenoweth et al., 1998), makes mtDNA a sensitive marker
for detecting patterns of genetic structure in natural systems. A mitochondrial genome
is considered as haplotype (Cavalli-Sforza, 1998). The haplotype diversity allows us
to determine variation among individuals within a population and among populations
(Billington, 2003).
Restriction Fragment Length Polymorphisms (RFLP) combined with
Polymerase Chain Reaction (PCR) technique has become a powerful tool to extract
haplotypic frequency data from mitochondrial DNA (Bernatchez & Danzmann,
1993). Using this technique, I attempt to evaluate level of mitochondrial DNA
variation within and among populations of cultured broodstock of L. calcarifer in
 3

Thailand compared to wild populations. This information may help the management
of genetic diversity of existing programs as well as the development of a selective
breeding program.

1.2. Objectives
1. Estimate genetic variation within hatchery and wild populations of L.
calcarifer using PCR-RFLP of the D-loop region of mitochondrial DNA.
2. Examine population differentiation among hatchery and wild populations.

1.3. Contribution to Knowledge

1. The genetic data will be useful in managing existing genetic diversity in
hatchery populations of L. calcarifer in Thailand.
2. These data should be useful for breeders and government agencies in
order to develop a selective breeding program for L. calcarifer.
3. Data for wild populations will provide basic information to aid
conservation efforts of native genepool of L. calcarifer in Thailand.

1.4. Scope of Study

This study will focus on the analyses of the D-loop region of mitochondrial
DNA to obtain genetic variation within and among hatchery, and wild populations
located around the Gulf of Thailand. The selected hatchery populations represent
important broodstock supplying fingerlings for Thailand.
In this study, I will use the RFLP technique with seven restriction enzymes
that cut at specific sites within the mtDNA D-loop region. Polymerase Chain Reaction
(PCR) will be used to amplify partial fragment of the D-loop region from extracted
genomic DNA. For each individual, restriction fragment patterns generated all
enzymes will be grouped into as composite haplotype. Haplotypic frequencies for
each population will be used for further analysis.
 4

1.5. Hypothesis
1. Level of genetic variation within hatchery populations of L. calcarifer is lower
than that of wild populations.
I hypothesized that broodstock used in L. calcarifer hatcheries have
lower genetic variation than the wild populations as consequence of
aquaculture practices. Aquaculture practices that may lead to the reduction of
genetic variation include using limited number of mating parents and
employing mass spawning leading to unequal sex ratio and unequal
contribution of each family (Miller & Kapuscinski, 2003; Frost, Evans, &
Jerry, 2007). Reduced genetic variation has been observed in hatchery stocks
relative to the wild population in fish species such as Oreochromis niloticus
in Cameroon (Brummett et al., 2004), Common carp, Cyprinus carprio in
Vietnam (Thai, Burridge, & Austin, 2007), Atlantic salmon, Salmo salar in
northern Spain (Horreo et al., 2008).

2. Genetic differentiation between wild L. calcarifer populations is high, but the

differences among hatchery populations are low
Lates calcarifer is catadromous fish, spawn in estuaries and moving
vertically to feeding ground in the upper part of river (Moore, 1982). Based on
a tagging and recaptured study, Russel & Garrett (1988) reported that
barramundi can move to another river up to 17 km along north-eastern
Australian from their origin habitat. A study employed the allozyme technique
revealed significant genetic differences between areas separated by about 150
km (Salini & Shaklee, 1988).
In absence of recombination, mtDNA should be more sensitive than
nuclear DNA in detecting differentiation between populations because the
mtDNA has smaller effective population size (Ne). Small Ne leads to high rate
of reduction of genetic variation within population due to genetic drift, this
process will allow for genetic divergence of disconnected populations
(Allendorf & Luikart, 2007).
A linear regression of genetic distances inferred from mtDNA
sequence (Tamura-Nei distance) and from microsatellite data (delta mu
 5

squared genetic distance), and geographic distance by Mantel test showed

positive correlation of the genetic distance with coastal distance (Figure 1.1;
Marshall, 2005). Based on mtDNA data, I hypothesized that the level of
genetic differentiation between two wild populations (Chantaburi and Nakhon
Si Thammarat) will be high because of its geographical distance (480 km a
part measuring as straightline across South China Sea, or about 900 km of
coastline distance).

Figure 1.1. Linear regression plot of Tamura-Nei’s and delta mu squared genetic
distance with coastal distance inferred from mtDNA sequencing (top)
and microsatellite (bottom) using Mantel test.

In contrast, I hypothesized that genetic differentiation among hatchery

populations are low. Based on informal interviews, hatchery managers prefer
introducing already domesticated broodstock from other fisheries
development centers or fish breeders to introducing wild individuals because
the convenience and lower mortality rate during transportation. This practice
will lead to genetic homogenization of various hatchery populations. Based
 6

on sequences of mtDNA control region and Single Strand Confirmation

Polymorphisms (SSC), Thai, Pham, and Austin (2006) suggested variation
among-population partitioned by AMOVA in Vietnamese common carp
(Cyprinus carpio) hatcheries was not significant (19.53% at P < 0.01). The
genetic homogenization among hatcheries populations was due to
interbreeding among lineages in order to produce genetically improved
strains.
 7

CHAPTER 2
LITERATURE REVIEWS

2.1. Biology of Asian seabass

Asian seabass, (Lates calcarifer Bloch, 1790) are large centropomid fish
(Actinoperygii: Perciformes: Centropomidae: Latidae). The body is elongate and
laterally compressed, with a relatively large mouth, slighty oblique upper jaw
extending behind the eye. The head profile is clearly concave. The lower edge of the
pre-operculum is serrated with a strong spine; the operculum has a small spine and a
serrated flap above the origin of the lateral line and the scales are ctenoid. The first
dorsal fin bears eight to nine spines and ten to eleven soft rays. The ventral fins have
spines and soft rays; the paired pectoral and pelvic fins have soft rays only; and the
caudal fin has soft rays and is rounded (Larson, 1999).
Classification of barramundi could be described as follows:
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Perciformes
Family: Latidae
Genus: Lates
Species: Lates calcarifer Figure 2.1. Morphology of Adult Seabass /
Barramundi (Lates calcarifer Bloch)

Source:www. fishase.org.

Based on recorded movement of tagged fish, Asian seabass or Barramundi is

categorized as a catadromous-demersal teleost fish (Moore, 1982), inhabiting coastal
areas, particularly estuaries and rivers (Russel & Garrett, 1988) with depth range 10-
40 m (Larson, 1999). In Papua New Guinea, larvae and young juveniles live in
brackish temporary swamps associated with estuaries, and after 2-3 years they move
into inland waters. After their third or fourth age, the fish migrates from inland to
coastal waters (Moore & Reynolds, 1982). Based on tagging and recaptured study,
movement of barramundi in north-eastern Australian coastline reached maximum 70
 8

km from estuaries to upstream the river as vertical migration, and maximum 17 km to

another river along coastline from their origin habitat (Russel & Garrett, 1988).
The von Bertalanffy growth parameters for infinitive length (L∞) and growth
(K) in L. calcarifer varies between populations. In Mary River and West Alligator
River, both located at Van Diemen Gulf, Australia, L∞ was estimated as 1374 mm and
996 mm with K = 0.148 and 0.216, respectively (Davis & Kirkwood, 1984). In
Fitzroy River, Queensland L∞ = 690 mm, growth K = 0.53 and t 0= 0.003 years (Stuart
and McKillup, 2002). Although there was variation of L∞ between river populations
in Northern Australian coastline, but the variation was not significant. Furthermore,
mean length at age 1 to 5 years among river populations was not statistically different
(Davis & Kirkwood, 1984).
L. calcarifer is sexually protandrous hermaphrodites; individual is sexually
matured as male in the first time. The gonad structure then develops as function as
female at the following ages (Moore, 1979). Length at first maturity in males was
observed at 250 mm in the Gulf of Carpentaria, Australia (Davis, 1982). In a wild
population of the Northern Territory and South-eastern Gulf of Carpentaria-Australia,
eggs can be optimally produced after 8 years old, therefore the proportion of mature
female in a population is very low due to limited numbers of survivor reaching that
age (Davis, 1982). However, the fecundity of L. calcarifer is very high (up to 46 x 106
eggs), so that the small proportion of females can maintain number of recruitment in a
population (Davis, 1984). In sex-biased molecular marker such as mitochondrial
DNA, limited number of females and high fecundity may result in low genetic
variation in a population.
The adult exhibits a single annual reproductive period, but the peak period
varied among areas. In French Polynesia sea cages, a breeding season starts from
October to February beginning with the dry and wet seasons (Guiguen et.al., 1994). In
Van Diemen Gulf - Northern Australia, the fish spawned from September to February
and in Gulf of Carpentaria, it starts from November to March (Davis, 1985).
 9

2.2. Ecology and Distribution

Adults of Seabass are carnivorous, but juveniles are omnivorous
(Sirimontaporn, 1988). In Seabass larval rearing, juveniles can consume several
zooplankton species including rotifer (B. rotundiformis), Artemia nauplii and water
flea (Moina sp.) (Pechmanee, 1997). For European seabass, Dicentrarchus labrax,
diet of 0-group mainly consist of crustaceans with the most important food organisms
including Decapoda, Mysidacea, Isopoda (Cabral & Costa, 2001) and Amphipods
(Laffaile et al., 2001). Feeding activity increased during summer. Short-term
variations were related to the period of the day and tidal cycle (Cabral & Costa,
2001).
Distribution of L. calcarifer spread along the Indo-West Pacific coasts:
eastern edge of the Persian Gulf to China, Taiwan and southern Japan, southward to
southern Papua New Guinea and northern Australia (Figure 2; Larson, 1999). A study
based on microsatellite and cytochrome b of the mtDNA data data supported that L.
calcarifer in the coastline of Australia originated from Indonesia, migrated to Western
Australia, then spread east and west along the coastline (Marshall, 2005).

Figure 2.2. World geographic distribution of Asian Seabass (Retrieved from

FIGIS-FAO, http://www.fao.org/figis/)
 10

2.3. Genetic processes in populations

Principally, genetic composition in nature changes continuously as respond
to the environmental change. In a long term, genetic changes will lead to two
consequences: survival adaptation and extinction. In a shorter term, genetic changes
affect population’s characteristics and demography (Avise, 2004). There are four
major microevolutionary processes that could change population genetic
characteristics, i.e. mutation, genetic drift, gene flow and natural selection (Allendorf
and Luikart, 2007).

• Mutation
The ultimate source of genetic variation in populations is mutation. There
are two major types of mutations: point (gene) mutations and chromosomal mutations.
Point mutation is a change in one nucleotide or several nucleotides in a single gene.
The change could be due to base pair substitutions, insertion or deletion.
Chromosomal mutation is a change in the number of chromosome or gene
arrangement in chromosomes (Hallerman & Epifano, 2003). In the maternally
inherited haploid DNA such as mitochondrial DNA (mtDNA) where recombination
does not occur, most of genetic variation comes from mutation events, particularly
point mutations that creating or deleting nucleotide(s) in mtDNA sequences (Avise,
2004).

• Genetic drift
Genetic drift is the changes in allele frequency in a population in successive
generations due to a random process. The magnitude of genetic drift in a population
depends on the levels of deviation from an ideal population such as unequal number
of male and female breeders, variance in family size, and different number of parents
in successive generations (Hallerman, 2003).
The outcome of genetic drift can not be predicted because of the random
process. Effect of genetic drift, however, can be estimated through simulation and the
result is highly depends on population size (Ne). The impacts of genetic drift are more
obvious in small populations. Two major impacts on the genetic composition of small
populations are (1) change of allele frequency, and (2) lost of genetic variation
 11

(Allendorf and Luikart, 2007). Theoretically, mtDNA has fourfolds lower population
size than nuclear DNA. Thus, mtDNA is more susceptible to genetic drift effects than
nuclear DNA (Avise, 2004).
Factors influencing the level of genetic drift in a hatchery population may
include inappropriate aquaculture practices such as mating limited number of parents,
and employing mass spawning leading to unequal sex ratio and unequal contribution
of each family (Miller & Kapuscinski, 2003; Frost, Evans, & Jerry, 2007). Loss of
within-population genetic variation in a hatchery population is caused by several
processes such as genetic drift, inbreeding and extinction vortex. These processes can
lead to fitness reduction that driven to greater inbreeding and loss of variation. Using
adequate number of broodstock in an appropriate mating strategy will help breeders
avoiding loss of within population genetic variation in hatchery (Miller &
Kapuscinski, 2003). For example, Sriphairoj, Kamonrat and Na-Nakorn (2007)
simulated alternative mating strategies for broodstock Mekong giant catfish
Pangasianodon gigas in Thailand and suggested that mating equal number of
broodstock (28 mature male) and (28 mature female) with lowest the mean genetic
relatedness (rxy < 0.07) was able to prolong the reduction of genetic diversity for short
term mating plan. In the same study, minimal kinship approach in the first generation
then followed by a random mating scheme with Ne larger than 30 individuals is
recommended to preserve genetic variation at least 90% for long term plan (over 100
years).

• Gene flow
Gene flow (or migration) is any movement of alleles from one population to
another. Those alleles recombine with local alleles through sexual reproduction.
Genetic interactions between two or more populations through gene flow will increase
or maintain genetic variability within a population, but will decrease genetic
distinctiveness among populations (Gharret & Zhivotovsky, 2003). In mitochondrial
DNA, gene flow can be indicated by haplotypes shared between two or more
genetically related populations (Avise, 2004).
There are two major factors governing gene flow in nature population:
intrinsic and extrinsic factors. Intrinsic factors cover the role of biological aspects of
 12

the species such as reproductive system (e.g. asexual reproduction, autogamy,

outcrossing, and ploidy), behavior and dispersal (e.g. gametic or zygotic dispersal,
gender differences and breeding behavior), and historical processes, such as historical
events in a populations. Extrinsic factors are including physical barriers, and
environmental selection factors determining the survival of a particular species
(Lowe, Harris and Ashton, 2004). Gene flow also can be resulted by human activities
such as artificial culture including restocking, marine ranching and fish escaped from
culture system (Gharret & Zhivotovsky, 2003).
The movements of broodstock among hatcheries will increase or maintain
within-population genetic variation, but will reduce among-population genetic
variation (or population identity). Based on AMOVA, Thai, Pham and Austin (2006)
indicated significantly high genetic variation within hatchery than among hatchery
populations (80.47% and 19.53, respectively; P<0.01) of common carp in Vietnam as
result of genetic improvement dissemination program that facilitate gene flow among
hatcheries. Lack of genetic divergence among white shrimp Litopenaeus vannamei
hatcheries in Brazil also has been identified as results of the gene flow effects due to
shared ancestry of initial founder composition of broodstocks used in all hatcheries
(Freitas, Calgaro and Galetti, 2007). To prevent genetic identity among hatcheries,
establishment of genetic improvement programs through broodstock’s interchange
among hatcheries is needed. Genetic lineage, life history patterns and ecology of
originating environment data of broodstock is important to increase the adaptive
chances of hatchery to the introduced broodstock (Miller and Kapuscinski, 2003).

• Natural selection
Genetic processes such as mutation, genetic drift and gene flow are can
cause change overtime, but natural selection is the primary process of adaptive
evolution. Natural selection is the process which favorable heritable traits become
more common and unfavorable heritable traits become less common in successive
generations due to differential survival or reproduction of phenotypes in a population
(Haliburton, 2004). Since phenotypes are highly associated with genotypes, unequal
probability of alleles survive or reproduce the future generation will determine their
allele frequency in a population (Allendorf, 2007, pp. 171-196).
 13

There are three modes of selection which alternatively change the genetic
composition at the end of population’s distribution: (1) Directional selection, that
produces one favorable phenotypes or fixed homozygote (either dominant or
recessive) outcome, (2) Stabilizing selection or normalizing selection, that favors
intermediate phenotype or fixed heterozygote outcome, and (3) Disruptive selection,
that favors extreme phenotypes or both dominant and recessive homozygote
(Hallerman, 2003). Outcome of three modes of selection can be illustrated as Figure.
2.3.

A B C
Figure 2.3. Outcome of three modes of selection: (A) Directional selection, (B)
Stabilizing selection, and (C) Disruptive selection (Hallerman, 2003).

2.4. Mitochondrial DNA as Genetic Marker for Population genetic

Analyses
Genetic variation in natural populations has been studied by using various
markers following the development of molecular techniques. Chromosome and
protein electrophoresis were commonly used for genetic variation studies because of
its fast and simplicity to observe polymorphisms and heterozygosity to estimate level
of variation (Allendorf & Luikart, 2007). Development in molecular techniques
allows us not only to infer variation from nuclear DNA but also cytoplasm DNA such
as mitochondrial DNA (mtDNA), chloroplast DNA, and ribosomal DNA. The
variation can be obtained from direct sequence and indirect methods to infer
nucleotide sequences including Restriction Fragment Length Polymorphisms (RFLP),
Amplified Fragment Length Polymorphisms (AFLP), Random Amplified
Polymorphic DNA (RAPD), Single-Strand Conformational Polymorphisms (SSCP),
DNA Microarrays and Microsatellites (Halliburton, 2004). Nuclear DNA and mtDNA
 14

contain rich information for variation studies on animal variation, particularly for fish
species (Billington, 2003; Brown & Epifano, 2003).
Animal mtDNA is a relatively small circular molecule of 15 to 20 kb,
comprising of 37 genes. These genes code for 22 tRNAs, 2 rRNAs, and 13 mRNAs.
The mRNA is protein coding involved in electron transfer and oxydative
phosphorilation in the mitochondria. The genome lacks introns and contains small
intergenic spaces in which the reading frames sometime overlap. The control region is
the only major non coding area of the mitochondrial genome. It is approximately 1 kb
in size, involved in the regulation and initiation of replication and transcription of the
mitochondrial genome (Terzioglu & Larson, 2007).
The use of mtDNA in recent years has been increasingly popular, especially
in phylogenetic and population genetic studies. Analysis techniques for mtDNA have
evolved from the use of restriction enzymes to detect differences in nucleotides of the
whole mtDNA genome (Lansman et al, 1981), to the use of polymerase chain reaction
(PCR) to amplify a particular region of mtDNA (Kocher et al., 1989). Characteristics
of mtDNA such as uniparental inheritance (Birky, Fuerst, & Maruyama, 1989),
smaller effective population size than nuclear DNA, and higher mutation rates
(Chenoweth et al., 1998), make mtDNA a sensitive marker for detecting patterns of
genetic structure in natural systems.
Different regions of mtDNA may be appropriate for different applications
because of the varied mutation rates among regions. For example, cytochrome b and
16S rDNA are suitable for describing phylogenetic relationships (Irwin, Kocher &
Wilson, 1991), while control region is appropriate for population genetic studies
(Brown et. al., 1986) because of its hypervariable nature. The use of PCR-amplified
mtDNA control region has been commonly used for genetic variation studies at
population levels, for example Asian Nile and red hybrid tilapia (Romana-Eugia et al.,
2004), Asian seabass or barramundi in Australia (Marshall 2005), and common carp
in Vietnam (Thai, Pham and Austin, 2006).
 15

2.5. Molecular Techniques for mtDNA Analysis

Mitochondrial DNA analysis requires molecular techniques to obtain
information related to nucleotide variation and its relative changes at an individual or
population level. Principally, there are two techniques commonly used for
mitochondrial DNA analysis, i.e. restriction fragment length polymorphisms (RFLP)
and direct sequencing. Both restriction pattern analysis and direct sequencing could be
performed on whole mtDNA or particular region within mtDNA genome.

2.5.1. Restriction Fragment Length Polymorphisms (RFLP)

Restriction Fragment Length Polymorphisms of a DNA genome can be
obtained using one or more restriction endonucleases. Fragments can, then, be
observed by electrophoresis gel separation based on their molecular weight. Variation
in the RFLP patterns may result from nucleotide substitution within restriction sites,
insertions or deletions of DNA, or sequence rearrangements (Avise, 2004, 67-78).
RFLP technique is commonly used in aquaculture genetics studies (Liu &
Cordes, 2004). RFLP method can be applied on whole mtDNA genome or a specific
region such as control region or cytochrome b. RFLP of a whole mtDNA genome is a
powerful tool to provide a comprehensive map for mtDNA variation e.g., mtDNA
diversity in wild and culture population of Brycon opalinus in Brazil (Hilsdorf et al.,
2002). However, RFLP for mtDNA genome needs large quantity and high quality of
purified mtDNA, involving methods that use either radioisotopes or complex
biochemical material.
Alternatively, RFLP of a specific region through Polymerase Chain Reaction
(PCR) offer a rapid, simple, and low cost method to detect polymorphisms (Kocher,
1992). This method provides vast utility in genetic variation study for both nuclear
and organelles DNA (Liu & Cordes, 2004). Furthermore, sampling for genetic
markers that are assayed using PCR-based technology can be carried out using small
amount of samples (e.g., scales or 10-3 g of tissue) so that lethal sampling can be
avoided (Scribner et al., 1998).
PCR amplification from a specific region requires appropriate primers.
Bernatchez and Danzmann (1993) designed LN20 and HN20 for amplification of ~1
kb control region of brook charr Salvelinus fontinalis Mitchill. These primers can also
 16

be applied in several fish species such as scad mackerel Decapterus russelli (Arnaud,
Bonhomme, & Borsa, 1999), Nile and hybrid tilapia (Romana-Eugia et al., 2004),
Moroccan sardines Sardina pilchardus (Atarhouch et. al., 2006) and Asian seabass
Lates calcarifer (Marshall, 2005) to seek population structure and differentiation.
MtDNA assay using the PCR-RFLP technique produces high sensitivity in
population genetic study. Bernatchez and Danzmann (1993) compared the level of
congruence of RFLP and sequencing data in describing genetic diversity and
phylogenetic relationships among mitochondrial DNA haplotypes of wild and
hatchery populations of brook charr Salvelinus fontinalis Mitchill from Ontario. This
report showed high congruent between both techniques in terms of mtDNA variation
detected per number of nucleotide sampled and their ability to describe phylogenetic
relationships among haplotypes.
Basic assignment of RFLP method is constructed from presence-absence
matrix of restriction sites (Nei & Miller, 1990). A pattern of fragment produced after
enzyme digestion notated by a specific letter. A composite of fragment patterns
generated by a set of restriction enzymes is described as haplotype. Frequency of
haplotypes within samples is the key source for data analysis (Nei & Tajima, 1981).

2.5.2. Direct Sequencing

DNA sequencing is a biochemical method to determine the order of the
nucleotide bases: adenine (A), guanine (G), cytosine (C) and thymine (T) in a DNA
strand. Main purpose of this method is seeking variation differences of nucleotides
within particular or whole DNA genome. Direct sequencing method allows us to
detect a nucleotide base changes (or differentiation) due to insertion, deletion or
substitution between two individuals.
Two general protocols for DNA sequencing include (1) Radioactive end
label or Maxam-Gilbert method and (2) Anneal primer or Sanger method (Avise,
32
2004, pp. 98-100). Radioactive end label method uses radioactive chemical P to
label one end of a DNA strand. DNA is then cleaved at base-specific position using
four chemical reagents for four DNA subsamples (Maxam & Gilbert, 1977). Sanger
method uses the 2’, 3’-dideoxy and arabinonucleoside replacing normal
deoxynucleoside triphosphates that act as specific chain-terminating inhibitors of
 17

DNA polymerase (Sanger, Nicklen & Coulsen, 1977). Both methods using
polyacrilamide gel electrophoresis to separate fragments at corresponding nucleotide
sequence.
Recent advance for sequencing technology has shifted from flat –
polyacrylamide gel electrophoresis to capillary electrophoresis, a method that
measures electric current of ion conduction of nucleotide molecule passing through
the 1 nanometer pores. Electric current specific to a nucleotide charge then read by a
detector, and then translated and recorded by computerized machine (Ewing,
Wallingford & Olefirowicz, 1989). To compare DNA sequences between two or more
individuals, sequence data need to be edited and aligned so that differences between
sequences can be proceed to the next analysis.
Mitochondrial DNA sequence data has been widely used for population
genetic study in aquaculture species, for example, mtDNA control region sequence
data was very informative to determine the level of genetic diversity within and
among population of cultured and wild Vietnamese common carp Cyprinus carprio,
and relationships with common carp strains from China, Indonesia, Japan, Hungary,
and India (Thai, Pham, & Austin, 2006). The mtDNA control region sequence data
also powerful to trace evolutionary relationship among 30 fish species from 14
suborders that closely related with L. calcarifer taken from Australia and Singapore
(Lin et al., 2005).
Although sequencing has higher sensitivity in detecting nucleotide variation
than RFLP analysis, magnitude of variation and differentiation resulted from both
analysis are congruent. Bernatchez and Danzmann (1993) provided an evidence of
congruence in terms of amount of variation and nucleotide differentiation between the
control region sequence and restriction site variation in mtDNA control region of
brook charr (Salvelinus fontinalis). This evidence included: (1) there was only one
different sequence haplotype undetected by RFLP analysis if some of rare haplotypes
from RFLP analyses (of 27 haplotypes) are converted into the same number of
sequence haplotype (11 haplotypes), (2) the number of mutations per nucleotide
detected in sequence analysis was approximately twice that of the number detected
from RFLP analysis, but linear relationships between the number estimated through
 18

both methods showed strong positive relationship, suggested identical efficiency in

sequence and RFLP analyses to detect mtDNA variations.

2.6. Genetic diversity of Asian seabass and species with similar life
histories
Genetic diversity of Asian seabass (L. calcarifer) and other species with
similar life history has been studied for decades. Most studies has focused on L.
calcarifer populations in Australia, where L. calcarifer is highly abundant in the wild.
Genetic markers used to describe genetic diversity of L. calcarifer in various
geographic locations include allozyme, mtDNA and microsatellite DNA markers.
Genetic variation in wild populations of L. calcarifer is relatively high
compared with other species with similar life histories. Using allozyme analysis,
Salini and Shaklee (1988) observed high genetic variations in L. calcarfer populations
in Northern Australia (average expected heterozygosity (He) = 0.120±0.185)
compared to European seabass Dicentrarchus labrax populations in Mediterranean
Sea (He = 0.041 and He = 0.113±0.015) (Lemaire et al., 2000; Allegrucci, Fortunato,
& Sbordoni, 2007), and wild brown trout Salmo trutta populations from Sourge river
(He = 0.007) and Orb river (He = 0.053), both located in the Mediterranean drainage
basin (Poteaux, Berrebi, & Bonhomme, 2001).
MtDNA marker also indicated high genetic variation in wild populations of
L. calcarifer. Using mtDNA control region sequences, Chenoweth et al. (1988)
observed high genetic diversity in term of haplotype diversity (ĥ) in populations of L.
calcarifer in Northern Australia and Western Arafura Sea (ĥ = 0.763 – 0.933).
Similarly, Doupe, Horwitz and Lymbery (1999) detected high haplotype diversity (ĥ
= 0.711 - 0.897) in populations of L. calcarifer in Western-Northern Australia. The ĥ
values of L. calcarifer were higher than, for example, wild populations of Salmo
trutta in Danube drainage-Austria (ĥ = 0.356 – 0.642) inferred from mtDNA control
region sequences analysis (Weiss et al., 2000).
Using nuclear DNA microsatellite data, the level of genetic diversity,
represented as expected heterozigosity (He), of the wild populations of L. calcarifer
were found to be high in the Northern Australia (He = 0.518 – 0.728); and Thailand
 19

(He = 0.75) (Marshall, 2005; Zhu et al., 2006). Differences result in obtained by
different studies may be influenced by characteristic and sensitivity of marker used,
number individuals sampled, and/or genetic composition has changed overtime in
corresponding populations.
Genetic variation in hatchery populations is relatively lower than wild
populations. Using Microsatellite and mtDNA sequence method, Sekino, Hara and
Taniguchi (2002) observed lower genetic variation within population in hatchery (He
= 0.59-0.71; h = 0.692-0.798) than wild population (He = 0.75-0.76; h=0.998) of
Japanese flounders Paralichthys olivaceus. Genetic variation study in wild and
hatchery populations of brown trout (Salmo trutta) indicated significant differences of
within-population genetic variation (Hansen et al, 1997). In a hatchery population,
low variation within population may indicate occurrence of genetic drift and
inbreeding (Kapuscinski and Miller, 2003). In wild populations, low genetic variation
may be facilitated by the lack of migration or gene flow, founding effect or bottleneck
process in the past (Allendorf & Luikart, 2007). Genetic variation in hatchery
population, however, can be higher than wild population. In some cases, Zhu et al.
(2006) observed the hatchery/stock I were slightly higher (He = 0.76) than wild
population because it consisted of individuals from genetically distinct sources.
Strong population structure among wild populations of L. calcarifer has
been indicated by high percentage (most values significant at P<0.001) of variation
among population (analyzed by analysis of molecular variance /AMOVA), clear
clustering pattern based on genetic distant (high distribution or FST supportive values
on a node), or exact test (Table 2.1). Recent study in wild populations of L. calcarifer
in Northern Australia (Marshall, 2005) showed a significant variation differences (P <
0.001) among river populations within province (percentage of variation = 28% and
61% from mtDNA and microsatellite, respectively) and among provinces (percentage
of variation = 3.1% and 47% from mtDNA and microsatellite, respectivelly). High
variation among populations and among groups indicated the existence of population
structure of L. calcarifer in Northern Australia. A PCR-RFLP study in the mtDNA of
brown trout Salmo trutta also indicated existence of population structure because of
high percentage of variation (88.83%; P<0.0001) among five lineage groups: Atlantic,
Danubian, Marmoratus, Mediterranean, and Adriatic (Bernatchez, 2001).
 20

The presence of population structure in L. calcarifer populations in Northern

Australia likely indicated stepping-stone migration model (Salini and Shaklee, 1988;
Marshall, 2005), where populations within group are genetically similar. The stepping
stone model is a spatial model of population structure where adjacent populations are
the primary sources of genetic material. Adjacent populations would them be more
genetically similar compared to more distant populations (Gharrett & Zhivotovsky,
2003).
Linear stepping-stone model of migration may fit to describe L. calcarifer
movement in the wild, because they migrate along the river in one direction, out to
coastal areas, and return to original rivers. This migration pattern will preserve the
within population genetic diversity (Marshall, 2005). A similar genetic diversity
pattern also has been observed in brown trout Salmo trutta populations within five
lineage groups (Atlantic, Danubian, Marmoratus, Mediterranean, and Adriatic), where
populations within a lineage group were more genetically similar compared to
populations in different lineage. The lower variation among populations within
lineages than among lineages (8.67% and 88.83% at P<0.0001 for the levels of
variation within and among lineages, respectivelly) not only indicated a one-
dimensional migration but also the existence of evolutionary lineages as a result of
geographic isolation during Pleistocene period (Bernatchez, 2001).
 21

Table 2.1. Genetic diversity of L. calcalifer and species with similar life histories
Species - Habitat Marker/ Number of Number Haplotype Nucleotide Partitioned Variation Reference
Technique Enzymes of diversity ( ĥ )/ diversity
used (for Haplotype Within Among Among group
Expected
RFLP) populations populations (%)
Heterozygosity
(%) within group
(He)
(%)
Lates calcarifer- Allozyme - - He Mean FST
Northern Australia 0.120±0.185 8.7 * Salini & Shaklee, 1988
( P < 0.001)
Lates calcarifer mtDNA/ - AMOVA, Haplotype based
Northern Australia Sequencing ĥ
- Coral Sea 17 0.763±0.044 0.0296±0.0054 8.6 10.4
- Gulf of Carpentaria 22 0.915±0.014 0.0369±0.0055 Chenoweth, et al.
- Western Arafura sea 24 0.933±0.012 0.0598±0.0093 ( P < 0.001) (P < 0.001) (1998)

AMOVA, Sequence based

3.5 30.3
(P = 0.009)
(P = 0.002)

Lates calcarifer mtDNA/ ĥ AMOVA

- Darwin (D) Sequencing 4 0.711±0.239 0.006±0.001 52.6 Doupe, Horwitz, &
- Fitzroy river (F) 17 0.897±0.045 0.017±0.006 (P < 0.001) Lymbery (1999)
- Ord river (O) 25 0.836±0.057 0.040±0.006
Lates calcarifer- mtDNA/ - - - AMOVA, mtDNA
Northern Australia Sequencing 10.9* 28.0* 61.1*
Marshall (2005)
Nuclear DNA/ - He - AMOVA, Microsatellite
Microsatellite 0.518(±0.073) – 49.8* 3.1* 47.0*
0.728(±0.067) All at P < 0.001
 22

Lates calcarifer- Nuclear DNA/

Singapore Microsatellite - He FST
- Wild (Thailand) 0.75 30
- Stock I 0.76 (P < 0.05) Zhu et al. (2006)
- Stock II 0.72
Dicentrarchus labrax Nuclear DNA/ - - He - FST Allegrucci, Fortunato,
Mediterranean Sea Allozyme 0.113±0.015 34 & Sbordoni (1997)
( P < 0.05)
Salmo trutta/ mtDNA-ND-1 6
Denmark ND-5/ND6/ 5 ĥ Hansen et al. (1997)
- Wild RFLP 11 0.764±0.104
- Hatchery 9 0.572±0.173
Salmo trutta/ mtDNA- 6 AMOVA
Mediterranean Sea Control ĥ 8.67* 88.83*
- Atlantic lineage region/ RFLP 0.582(±0.011) 0.0020 62.42 (P < 0.0001) (P < 0.0001) Bernatchez (2001)
- Danubian lineage 0.931(±0.005) 0.0045 78.48
- Marmoratus lineage 0.851(±0.009) 0.0018 93.73
- Mediterranean lineage 0.523(±0.011) 0.0005 75.07
- Adriatic lineage 0.511(±0.007) 0.0005 90.42
Salmo trutta
Mediterranean Sea
He
Sorgue drainage Nuclear DNA/ 0.0007 - 0.087 - - - -
Orb drainage Allozyme 2 0.0532- 0.076
ĥ Poteaux, Berrebi, &
Sorgue drainage mtDNA- 0.06 -0.25 Bonhomme (2001)
Orb drainage control region 0.20 -0.49
/ RFLP
He
Sorgue drainage Microsatellite 0.4- 0.73
Orb drainage 0.59-0.84
 23

2.7. Genetic Data Analysis

Analysis of genetic data is crucial for population genetic studies. The aim of
genetic data analysis is to provide objective, stable classification of individuals into
groups (Shaklee and Currens, 2003). Principally, there are two mathematical models
to characterize population genetics in terms of RFLP technique data, i.e. genetic
diversity within populations and genetic differentiation among populations (Nei and
Tajima, 1981).

2.7.1. Genetic diversity within populations

Genetic variation in wild population reflects the occurrence of evolutionary
events, such as population bottlenecks, and presence of population structure (Avise,
2004, 478-515). In aquaculture populations, low genetic diversity within population
reflects the impacts of genetic drift or aquaculture practices that lead to reduction of
genetic variation (Billington, 2003). Populations with high genetic variation within
population may have high potential to adapt to environmental changes or disturbance
(Nguyen et al., 2006a).
Two important calculations for describing genetic diversity within
populations, are haplotype diversity and nucleotide diversity (Billington, 2003).
Haplotype diversity (ĥ), or gene diversity is subject to random variation due to stochastic
changes of haplotype frequencies. This can be estimated by the Nei and Tajima method
(1981; Equation 1).

………………………(1)

where n is the number of sample in a group, xi is the frequency of ith haplotype

composite and l is the number of haplotype composite. Haplotype diversity is
analogous to expected heterozygosity for nuclear, codominant DNA markers.
Nucleotide diversity ( ) is the average of nucleotide differences per site
between two randomly choosen DNA sequences (Nei and Li, 1979), and can be
estimated by Equation 2;

…………………………(2)
 24

where xi and xj are the frequencies of ith and jth haplotype in a population, πij is the
difference between haplotype i and j, and n is the number of individuals in the
population (Nei and Tajima, 1981). Nucleotide diversity will indicate haplotype
variation within populations. Using the Arlequin program, calculations of haplotype
and nucleotide diversity could be performed simultaneously (Excoffier, Laval, &
Schneider, 2005).

2.7.2. Genetic Differentiation among Populations

Genetic differentiation is determined from nucleotide differences between
individuals or populations. Analysis of genetic differentiation among populations
provides information in genealogy at a microevolutionary scale such as parentage and
kinship, historical population size, geographic distribution and gene flow (Avise,
2004, pp. 491–497). In aquaculture populations, genetic differentiation among
populations analysis is very useful for identifying populations, assessing gene flow
and identifying its potential origin, and designing selection criteria of broodstock
(Miller and Kapuscinski, 2003). Levels of genetic differentiation including estimation
of within population, among populations within groups, and among populations
among groups.
Genetic differentiation at a population level relies upon haplotypic frequency
differences inferred from several restriction site patterns. A method for assessing
genetic differentiation between populations was proposed by Nei and Tajima (1981);
it is analogous to genetic differentiation in sequence data. Consider pi and qi as
frequency of the ith haplotype in population X and Y, respectively, mean number of
genetic differentiation between two randomly chosen haplotypes ( , one each

from populations X and Y can be described as follow:

………..……………………(3)

Where vij = restriction site difference between haplotype i and j. If the sample
frequencies of the ith haplotype are xi and yi in population X and Y, respectively, the
value in Equation (3) can be estimated with Equation (4):

…………….………………(4)
 25

This estimation, however, assumes that there are polymorphisms of the restriction site
differences within populations, and the amount of restriction-site differences within
populations as described by equation (3) and equation (4). If populations to be
compared are closely related, the assumption will not be valid. Thus, the number of
net restriction site differences, or genetic differences should be subtracted from the
total differences among population, can be estimated by Equation (5):

………..………………………….(5)

where and are the values of described in Equation (4) in population X and Y,

respectively (Nei and Li, 1979).

Genetic differentiation also can be indicated by Φ-statistics developed by
Excoffier, Smouse & Quattro (1992) by calculating ΦST, ΦSC, and ΦCT, as the
correlation of random haplotypes among groups, among populations within a group,
and among individuals within a population, respectively. These equations can be
described as follows:

ΦST ΦSC ΦCT ……..(6)

where , , and are the associated covariance component for groups,

populations within a group, within populations, and total variation, respectively.
These calculations can be performed by Analysis of Molecular Variance provided in
the Arlequin program (Excoffier, Laval, & Schneider, 2005), an analogous to
Analysis of Variance (ANOVA) of a variable. AMOVA partitions existing genetic
variation into different hierarchical levels (Table 1).

Table. 1 General design for hierarchical analysis of molecular variance (AMOVA).

Source of variation d.f MSD Expected MSD
Among groups G–1 MSD/(AG)
Among populations within groups MSD/(AP/WG)
Among individual within populations MSD/(WP)
Total N–1
Note: MSD = Mean squared deviations ; G = number of group ; Ig = number of populations in g group ; N
= number of overall sample ; n, n’, n’’ = average sample size of particular hierarchical level as described
at Excoffier et.al, (1992)
 26

Permutation analysis method can be used for testing the significance of the
variance component ( , , and ) analysis and Φ-Statistics (Equation 6) to
obtain the null distribution and test for the significance of among groups (ΦST and
); among populations within group (ΦSC and ); and among individual within
populations (ΦSC and ). This method requires fewest assumptions, that is null
hypothesis, where each samples assumed as part of global population and variation
was due to random sampling in the structure of populations (Excoffier, Smouse and
Quattro, 1992).
The mtDNA restriction fragment data can be used to measure genetic
distance based on relatedness estimation between populations. Upholt (1977) first
described the relationship between the proportion of fragments shared in mtDNA-
restriction fragments and nucleotide sequence divergence (Avise, Lansman, & Shade,
1978). If Nx and Ny are the number of restriction fragments observed in sequences X
and Y, and Nxy is the number of shared fragment in both corresponding sequences, the
overall shared fragments proportion (F) is calculated by the Equation (7):

…..……………………………….(7)

Fragment patterns produced from enzyme digestion can be used to estimate sequence
divergence between each pair of samples based on the percentage nucleotide
substitution (p), that is the proportion of fragments shared between two digested
sequences (F), and number of base pairs recognized by restriction enzyme (r) by the
following equation:
…………..(8)

Genetic distance is expressed from the proportion of nucleotide substation by

construct pairwise matrix between observed samples (Avise, Lansman, & Shade,
1978).

Genetic distance calculation based on Equation (8), however, requires the

number of base pair recognized by enzyme restriction (r), where this value is more
complex with recent development of multi-recognition restriction enzymes.
 27

Alternativelly, genetic distance can be derived by calculating pairwise FST values. The
FST values can be derived based on drift model, including mutation and other factors
affecting gene frequencies (Reynolds, Weir, & Cockerham, 1983) and Nei’s average
number of restriction site differences between populations (Nei and Li, 1979).
Reynold’s distance is defined as D = - log (1- FST), where

FST = 1 – (1-1/N)t = 1 – e-t/N………..……………(9)

where N is the initial size of reference population, and t is the number of known
generation from the initial population to the present population, assumed that the
reference population is randomly mating, completely isolated and constant in size.
Distances based on coancestry coefficient - the probability that two
homologous genes from two individual that are identical by decent - is designed to
measure the divergence FST between populations of size N that is caused by drift
during t generations ago. For short divergence times, the genetic distance D is
approximately proportional to t/N (Reynolds, Weir, & Cockerham, 1983).
Nei’s average number of restriction site differences between populations is
provided as Nei’s raw (D) and net ( DA). Nei’s raw (D) is the difference number of
restriction site between populations A and B, while Nei’s net is the average number of
net nucleotide substitutions per site (Nei & Li, 1979). The difference number of
restriction site between populations A and B ( ) can be calculated by Equation
(10):
………..(10)

and

……….…..……..(11)

where k and k’ are the number of distinct haplotypes in populations A and B,

respectively, xAi is the frequency of the i-th haplotype in population A, and δij is the
number of restriction site differences between haplotype i and haplotypes j.
Relationship tree among populations can be constructed using Neighbor-
Joining (NJ) Method (Saitou and Nei, 1987) based on pairwise genetic distance
values. The NJ method calculates and combines closely related number of operational
 28

taxonomic units (OTU’s) into a pair of neighbor. Each pair of neighbor, or tree
branch, is connected by a node, and length of branch is determined based on the
number of OTU of corresponding neighbor joined. Thus, distance between population
1 (DOTU-1) and population 2 (DOTU-2) is: D(1-2) = DOTU-1 + DOTU-2. For neighbor joining
of three populations or more, the distance between combined OTU of population 1
and population 2, and another OTU j can be written as Equation (12):

) ………………(12)

where D1j and D2j are the distance between OTU 1 and OTU j ; and OTU 2 - OTU j,
respectively. The small distance value indicates closely related pair. Usually first
draw of branch length started from those least values following by larger values.
Distance values, however, often more complicated to be constructed and have various
relationship possibilities. Hence, statistical method known as the ‘bootstrap’ should be
employed to assess reliability of tree construction by resampling the tree. Majority
consensus trees can be use to construct a tree inferred from majority of bootstrap tree
samples (Felsenstein, 1985).
 29

CHAPTER 3
RESEARH METHODOLOGY

3.1. Samples Collection

This study focuses on the mitochondrial DNA (mtDNA) variation of Asian
seabass populations along the Gulf of Thailand, where most of Asian seabass
aquaculture are located (Department of Fisheries of Thailand, 2006). Fin clips will be
collected from cultured and wild populations. Samples represented cultured
broodstock will come from three locations: Rayong Coastal Fisheries Reseach and
Development Center hatchery – Rayong (RA) , a private hatchery - Chonburi (CB)
and Nakhon Si Thammarat Coastal Fisheries Reseach and Development Center -
Nakhon Si Thammarat (NK), and the wild samples will come from two locations:
Chantaburi (CH) and Nakhon Si Thammarat (PN) (Figure 3.1).

Figure 3.1. A map of sampling locations: Chantaburi (CH), Rayong (RA) and
Chonburi (CB), Nakhon Si Thammarat (NK and PN). Circle and triangle
notation indicates cultured and wild sampling populations, respectively.
 30

Each samples consist of 39 - 67 individuals with size varied from 4 to 7

kilograms weight (Table 3.1). For cultured broodstock, small amount of caudal fin
clips will be cut without further harm to the fish. Wild samples will be collected from
local fisherman and fish distributors. Fin clips will be preserved with 95% ethanol.

Table 3.1. Population source and number of sample

Sample/Population Code Wild/ Number of
Cultured Individuals
Chantaburi CH Wild 55
Rayong RA Cultured 67
Chonburi CB Cultured 56
Nakhon Si Thammarat/Sichon NK Cultured 39
Nakhon Si Thammarat/Phak Nakhon PN Wild 65

3.2. DNA extraction

Total DNA will be extracted from the samples using a universal and rapid
salt-extraction protocol described by Aljanabi and Martinez (1997). A small piece of
fin clip will be submerged in 400 µl lysis buffer (0.4 M NaCl; 10 mM Tris-HCL pH
8.0; 2 mM ethylenediamene tetraaceticacid (EDTA) pH 8.0; 20% sodium dodecyl
sulfate/SDS (2% final concentration)) and 20 mg/ml Proteinase K (USBiological,
USA). The fin clip samples will then be crushed and incubated in waterbath at 55oC
overnight. Under this condition, SDS will digest cell membrane lipid layer for cell
lysis, and proteinase K will be at optimum condition for protein digestion.
After protein digestion, DNA can be separated from cell debris by adding 6
M NaCl to each sample tube, and centrifugation at 10,000 rotate per minute (rpm) for
30 minutes. About 450 µl of the supernatant containing DNA to be transferred into a
new tube. DNA is precipitated using equal volume of 450 µl Isopropanol, then
incubated at -20oC for an hour and followed by centrifugation at 10,000 rpm for 20
minutes. Supernatant will be quickly poured. The salt remains will be washed with
1000 µl 70% Ethanol, and spun at 10,000 rpm for 20 minutes. After supernatant is
poured, the pellet containing DNA is then dried for an hour. Dried DNA pellet is
resuspended in 50 µl 0.1 X TE.
 31

To quantify extracted DNA, about 2µl of the DNA and 1 µl of loading dye is
then electrophoresed in 1 % Agarose gel with dimension of the gel is 15 x 10 cm, and
2x20-well of 1.5 mm fixed-height comb (BIO-RAD SubcellGT, Italy). The mobility
of extracted DNA was compared with 1 kb size standard and λ (lambda) DNA
(Invitrogen, USA) with known quantity of 50 ng, 100 ng, and 200 ng. DNA
concentration will be use as reference in determining PCR ingredients.

3.3. PCR Amplification

Polymerase Chain Reaction (PCR) is used to amplify a 1 kb of the
mitochondrial control region using primer LN20 (5’-ACC ACT AGC ACC CAA AGC
TA-3’) and HN20 (5’-GTG TTA TGC TTT AGT TAA GC-3’) designed by Bernatchez
and Danzzman (1993). A PCR reaction contains 1x PCR buffer, 0.4 mM MgCl2, 2
mM dNTPs, 2 pmole each primers, 0.5 U Taq Polymerase (Vivantis), 15-50 ng DNA
template and distilled water up to 20 µl of the solution. PCR is performed in a thermal
cycler (Hybaid Touchdown) for 30 cycles (92oC, 60 s; 50oC, 60 s; 72oC,90s). The
initial cycle is a 1 minute denaturing step at 92oC and followed by a 10 minutes final
extension at 72oC.
PCR products will be electrophoresed run in 1% Agarose (Vivantis) at 65 V
for 45 minutes. After which the gel will be stained in Ethidium bromide for 10
minutes. DNA fragments will be visualized on a UV transluminator (VILBER
LOURMAT ETX-40M, France).

3.4. Enzyme Digestion

As a preliminary assessment of potential restriction enzymes and restriction
sites for samples included in this study, seven individuals from Chantaburi (CH),
Rayong (RA) and Chonburi (CB) were sequenced to obtain potential restriction sites
of the mtDNA control region. PCR products were cleaned using RBC-Bioscience kit
following the manufacture instructions, and sequenced (ABI I.6.0, Macrogen Inc.
South Korea). Sequences were checked using Sequence scanner v1.0, compared with
sequences of L. calcarifer available in the GenBank database using BLAST
(http://blast.ncbi.nlm.nih.gov/Blast.cgi), alingned using BioEdit (Hall, 2001) and
 32

adjusted by eyes for accuracy. Restriction site analysis was determined using the
software Restriction Mapper (http://www.restrictionmapper.org). Sequences of L.
calcarifer obtained from the preliminary assessment had 96 – 97% sequence identity
to L. calcarifer sequences available in GenBank (accession number DQ012430.1,
DQ012429.1, DQ012425.1 and DQ012432.1.
Based on the sequence differences, I identified seven restriction enzymes
that would reveal distinct DNA patterns of mtDNA control region of L. calcarifer:
FauI (New England Biolabs, England), HindII, EcoRI, EcoRV, VneI, Bse1I and
BstENI (Vivantis, Malaysia) (Table 3.2). About 100 ng of PCR-DNA template will be
digested by 0.1 unit enzyme restriction in 10x buffer and water. The mixture then
incubated for 16 hour at 55oC for FauI; 37 oC for HindII, EcoRI, EcoRV, and EcoNI;
and 65oC for Bse1I and BstENI.

Table 3.2. Recognition size sensitivity of restriction enzymes used

Enzyme Cut position Site length Number Expected size of
(bp) of cut fragments (bp)*
FauI CCCGC(N)4↓ 5 1 439 & 332
GGGCG(N)6↑
HindII GTY↓RAC 6 1 513 & 258
CAR↑YTG
EcoRI G↓AATTC 6 1 414 & 357
CTTAA↑G
EcoRV GAT↓ATC 6 1 606 & 165
CTA↑TAG
VneI/ ApaLI G↓TGCAC 6 1 498 & 273
CACGT↑G
Bse1I / BsrI ACTGGN↓ 6 1 449 & 322
TGAC↑CN
BstENI/EcoNI CCTNN↓NNNAGG 6 1 436 & 335
GGANN↑NNNTCC
* Expected size of fragment obtained from L. calcarifer D-loop mtDNA from GenBank (accesion
number DQ012430.1).

Digested PCR-DNA will be loaded into 1.5 % Agarose and run at 65 V for
70 minutes. A 50 bp standard ladder (Invitrogen, USA) and ~1 kb undigested PCR
product will be used as measurement references. The gel will be stained in Ethidium
bromide for 10 minutes and washed in water. DNA fragment will be visualized on
UV transluminator (Vilber Lourmat, France) and photographed for manual
measurement and documentation.
 33

Size of DNA fragments (basepairs, bp) will be measured relative to standard

ladder and undigested PCR product size. Unique size and number of fragments of
each enzyme digestion for all samples will be notated with specific letter such as A,
B, C, etc. Size of fragment data will be used to construct binary data representing
present (1) and absence (0) fragment for analysis of nucleotide diversity. Notation of
fragment pattern data will be used to construct haplotype composite for the analysis of
haplotype diversity.

3.6. Data Analysis

Each pattern of fragment produced after a enzyme digestion will be assigned
to a specific letter representing each cut position. I construct a composit haplotype
based on combinations of unique patterns generated by each enzyme. Haplotype data
will be analyzed for genetic diversity within populations and genetic differentiation
among populations.

- Genetic Diversity Within populations

I will describe genetic diversity within populations in terms of haplotype
diversity and nucleotide diversity (Billington, 2003). Haplotype diversity (ĥ), or gene
diversity, can be estimated by Equation (1) in Chapter 2, while Nucleotide diversity
( ) can be estimated by of Equation (2) in Chapter 2 (Nei & Tajima,1981).
Calculations of haplotype diversity and nucleotide could be performed simultaneously
by Analysis of Molecular Variation (AMOVA) using computer programs Arlequin
(Excoffier, Laval, & Schneider, 2005) and GenAlex (Peakal and Smouse, 2006).
To test the homogeneity of haplotype frequencies between samples, I will
use program MONTE implemented in the Restriction Enzyme Analysis Package
(REAP) (McElroy et al., 1992). Significant level for multiple tests significance levels
will be adjusted by the sequential Bonferoni technique (Rice, 1989).

- Genetic Differentiation Among Populations

I will use two approaches to analyze genetic differentiation among
populations, analysis of molecular variation (AMOVA) and cluster analysis based on
genetic distances implemented in the Arlequin program (Excoffier, Laval, &
 34

Schneider, 2005). This routine will search variation partitioned to two levels: among
individuals within a population, and among populations within groups. Significance
level of the covariance components for among populations (σ2b), within populations
(σ2c), and total variation (σ2T) also will be tested using Φ-statistics and the P values
will be generated by 1000 random permutation in AMOVA.
Differences among populations may reflect the demographic history of a
population such as level of migration rate or gene flow. This data may detect
evolutionary event such as bottlenecks and founder events in the past (Avise, 2004).
Information of variation differences among population data can also be used for stock
identification in wild and hatchery populations, particularly in the broodstock
management program (Billington, 2003).
Neighbor-Joining tree for population relationships will be constructed based
on a Nei genetic distance matrix as described in Equation (12) of Chapter 2 (Saitou
and Nei, 1987). Program Neighbor from Phylogeny Inference Package (PHYLIP) can
be used to perform bootstrapping to test the reliability of clusters on the tree
(Felsenstein, 1985; 2008). Constructed consensus dendogram will be visualized on the
TreeView program (Page, 2001).
 35

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