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THESIS PROPOSAL
YUSMANSYAH
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
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.
1.5. 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).
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.
CHAPTER 2
LITERATURE REVIEWS
Source:www. fishase.org.
• 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
• 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).
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
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).
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
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
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)
3.5 30.3
(P = 0.009)
(P = 0.002)
………………………(1)
…………………………(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).
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,
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)
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
and
……….…..……..(11)
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
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
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.
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.
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
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
REFERENCE
Aljanabi, S.M., & Martinez, I. (1997). Universal and Rapid salt-extraction of high
quality genomic DNA for PCR-based techniques. Nucleic Acid Research,
25(22), 4692-4693.
Allegrucci, G., Gortunato, C., & Sbordoni, V. (1997). Genetic structure and allozyme
variation of seabass (Dicentrarchus labrax and D. punctatus) in the
Mediterraneean Sea. Marine Biology, 128 (2), 347 – 358.
Allendorf, F.W. & Luikart, G. (2007). Conservation and the Genetics of Populations.
Wiley-Blackwell Publisher.
Arnaud, S., Bonhomme, F., & Borsa, P. (1999). Mitochondrial DNA analysis of the
genetic relationships among populations of scad mackerel (Decapterus
macarellus, D. macrosoma and D. russelli) in South-East Asia. Marine
Biology, 135, 699 – 707.
Atarhouch, T., Rüber, L., Gonzalez, E.G., Albert, E.M., Rami, M., Dakkak, A., &
Zardoya, R. (2006). Signature of an early genetic bottleneck in a population
of Moroccan sardines (Sardina pilchardus). Molecular Phylogenetics and
Evolution, 39, 373 – 383.
Avise, J.C. (2004). Molecular markers, natural history and evolution, (2nd edition).
Sinauer Associates, Inc. Publishers, Sunderland, Massachussets.
Avise, J. C., Lansman, R.A., & Shade, R.O. (1978). The use of Restriction
Endonucleases to Measure Mitochondrial DNA Sequence Relatedness in
Natural Populations. I. Population Structure and Evolution in the Genus
Peromyscus. Genetics, 92, 279 – 295.
Bernatchez, L. (2001) The evolutionary history of brown trout (Salmo trutta L.)
inferred from phylogeographic, nested clade, and mismatch analyses of
mitochondrial DNA variation. Evolution, 55 (2), 351-379.
Bernatchez, L., & Danzmann, R. G. (1993). Congruence in control-region Sequence
and restriction- site variation in Mitochondrial DNA of brook charr
(Salvelinus fontinalis Mitchill). Molecular Biology and Evolution, 10(5),
1002-1014.
36
Cabral, H., & Costa, M.J. (2001). Abundance, feeding ecology and growth of 0-group
sea bass, Dicentrarchus labrax, within the nursery areas of the Tagus
estuary. Journal of the Marine Biological Association of the UK, 81(4),
679-682.
Cavalli-Sforza, L. L. (1998). The DNA revolution in population genetics. Trends in
Genetics, 14(2). 60-65.
Chenoweth, S.F., Hughes, J.M., Keenan, C.P., & Lavery, S. (1998). Concordance
between dispersal and mitochondrial gene flow: Isolation by distance in
tropical teleost, Lates calcarifer (Australian Barramundi). Heredity, 80, 187-
197.
Davis, T.L.O. (1982). Maturity and Sexuality in Barramundi, Lates calcarifer
(Bloch), in the Northern Territory and South-eastern Gulf of Carpentaria.
Australian Journal of Marine and Freshwater Research, 33, 529-545.
37
Doupe, R.G., Horwitz, P., and Lymbery, A.J. (1999). Mitochondrial genealogy of
Western Australian barramundi: applications of inbreeding coeffcients and
coalescent analysis for separating temporal population processes. Journal of
Fish Biology, 54, 1197-1209.
English L.J., Maguire G.B. & Ward R.D. (2000). Genetic variation of wild and
hatchery populations of the Pacific oyster, Crassostrea gigas (Thunberg), in
Australia. Aquaculture, 187, 283-298.
Esposti, M.D., Crimi, M., Ghelli, A., Patarnello, T., Meyer, A. and De Vries, S.
(1993). Mitochondrial cytochrome b: evolution and structure of the
protein". Biochemical and Biophysical Acta, 1143(3), 243–271.
Excoffier, L., Smouse, P. F., & Quattro, J.M. (1992). Analysis of Molecular Variance
Inferred From Metric Distance Among DNA Haplotypes: Application to
Human Mitochondrial DNA Restriction Data. Genetics, 131, 479-491.
Excoffier, L., Laval, G., & Schneider, S. (2005). Arlequin ver 3.1: An Integrated
Software Package for Population Genetics Data Analysis. Evolutionary
Bioinformatics Online, 1, 47-50.
38
Kocher, T.D. (1992). PCR, direct sequencing, and the comparative approach. PCR
Methods Application, 1, 217 – 221.
Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X.,
& Wilson, A.C. (1989). Dynamics of Mitochondrial DNA Evolution in
Animals: Amplification and Sequencing with Conserved Primers.
Proceedings of the National Academy of Sciences of the United States of
America, 86(16), 6196 – 6200.
Laffaile, P., Lefeuvre, J.C., Schricke, M.T., & Feunteun, E. (2001). Feeding Ecology
of 0-Group Sea Bass, Dicentrarchus labrax, in Salt Marshes of Mont Saint
Michel Bay (France). Estuaries, 24(1), 116-125.
Lansman, R. A., Shade, R. O., Shapira, J. F., & Avise, J. C. (1981). The Use of
Restriction endonucleases to measure mitochondrial DNA sequence
relatedness in natural populations; III. Techniques and Potential
Applications. Journal of Molecular Evolution, 17(4), 214-226.
Larson, H. (1999). Order Perciformes. Suborder Percoidei. Centropomidae. Sea
perches. p. 2429-2432. In K.E. Carpenter & V.H. Niem (eds.). FAO species
identification guide for fishery purposes. The living marine resources of the
Western Central Pacific. Volume 4. Bony fishes part 2 (Mugilidae to
Carangidae). FAO. Rome.
Lemaire, C., Allegrucci, G., Naciri, M., Bahri-Sfar, L., Kara, H., & Bonhomme, F.
(2000). Do discrepancies between microsatellite and allozyme variation
reveal differential selection between sea and lagoon in the sea bass
(Dicentrarchus labrax) ?. Molecular Ecology, 9, 457-467.
Le Vay, L., Carvalho, G.R., Quinitio, E.T., Lebata, J.H., & Fushimi, H. (2007).
Quality of hatchery-reared juveniles for marine fisheries stock enhancement.
Aquaculture, 268, 169-180.
Li, Q, Xu, K., & Yu, R. (2007). Genetic variation in Chinese hatchery populations of
the Japanese scallop (Patinopecten yessoensis) inferred from microsatellite
data. Aquaculture, 269, 211-219.
Lin, G., Lo, L.C., Zhu, Z.Y., Feng, F., Chou, R., & Yue, G.H. (2006). The Complete
Mitochondrial Genome Sequence and Characterization of Single-Nucleotide
41
Nei, M., & Li, W. H. (1979). Mathematical Model for Studying Genetic Variation in
Terms of Restriction Endonucleases. Proceeding of National Academy of
Science USA, 76(10), 5269-5273.
Nei, M., & Tajima, F.. (1981). DNA Polymorphism Detectable by Restriction
Endonucleases. Genetics, 97. 145-163.
Nei, M., & Miller, J.C. (1990). A simple method for estimating average number of
nucleotide substitutions within and between populations from restriction
data. Genetics, 125, 873-879.
Nguyen, T.T.T, Hurwood, D., Mather, P., Na Nakorn, U., Kamonrat, W., & Bartley,
D. (2006a). Manual on apllications of molecular tools in aquaculture and
inland fisheries management, Part 1: Concptual basis of population genetic
approaches. NACA monograph no. 1, 80 p.
Nguyen, T.T.T., Ingram, B., Sungan, S., Gooley, G., Sim, S.Y., Tinggi, D. & De
Silva, S.S. (2006b). Mitochondrial DNA diversity of broodstock of two
indigenous mahseer species, Tor tambroides and T. douronensis
(Cyprinidae) cultured in Sarawak, Malaysia. Aquaculture, 253, 259-269.
Nosil, P. (2008). Speciation with gene flow could be common. Molecular Ecology,
17(9), 2103-2106.
Page, R.D.M. (2001) TREEVIEW: An application to display phylogenetic trees on
personal computers. Computer Applications in the Biosciences, 12, 357-358.
Peakall, R., & Smouse P.E. (2006). GENALEX 6: Genetic Analysis in Excel.
Population Genetic Software for Teaching and Research. Molecular Ecology
Notes, 6, 288-295.
Pechmanee, T. (1997). Status of Marine Larviculture in Thailand. Hydrobiologia,
358(1-3), 41- 43.
Poteaux, C., Berrebi, P., and Bonhomme, F. (2000). Allozymes, mtDNA and
microsatellites study introgression in a stocked trout populations in France.
Reviews in Fish Biology and Fisheries, 10, 281-292.
Pourkazemi, M., Skibinski, D.O.F., & Beardmore, J. A. (1999). Application of
mtDNA d-loop region for the study of Russian sturgeon population structure
from Iranian coastline of the Caspian Sea. Journal of Applied Ichthyology,
15(4-5), 23 - 28.
43
Reynolds, J., Weir, B.S., & Cockerham, C.C. (1983) Estimation of the coancestry
coefficient: basis for a short-term genetic distance. Genetics, 105, 767-
779.
Rice, W.R. (1989) Analyzing Tables of Statistical Tests. Evolution, 43 (1), 223-225.
Romana-Eguia, M.R.R., Ikeda, M., Basiao, Z.U., & Taniguchi, N. (2004). Genetic
diversity in farmed Asian Nile and red hybrid tilapia stocks evaluated from
microsatellite and mitochondrial DNA analysis. Aquaculture, 236, 131-150.
Russel, D. J., & Garret, R. N. (1988). Movements of juvenile Barramundi, Lates
calcarifer (Bloch) In North-Eastern Queensland. Australian Journal of
Marine and Freshwater Researches, 39, 117 – 123.
Salini, J., & Shaklee, J.B. (1988). Genetic structure of Barramundi (Lates calcarifer)
stocks from Northern Australia. Australian Journal of Marine and
Freshwater Research, 39, 317-329).
Sanger, F., Nicklen, S., & Coulson, A.R. (1977). DNA sequencing with chain-
terminating inhibitors. Proceedings of the National Academy of Sciences of
USA 74 (12). 5463-5467.
Scribner, K.T., Crane, P.A., Spearman, W.J., & Seeb, L.W. (1998). DNA and
allozyme markers provide concordant estimates of population
differentiation: analyses of U.S. and Canadian populations of Yukon River
fall-run chum salmon (Oncorhynchus keta). Canadian Journal of Fisheries
and Aquatic Science, 55, 1748 – 1758.
Sekino, M., Hara, M., & Taniguchi, N. (2002). Loss of microsatellite and
mitochondrial DNA variation in hatchery strains of Japanese flounder
Paralichthys olivaceus. Aquaculture, 213, 101-122.
Shaklee, J.B., & Currens,K.P. (2003). Genetic stock identification and risk
assessment. Pages 291-328 in E. M Hallerman (editor). Population genetics:
Principles and applications for fisheries scientist. American Fisheries
Society. Bethesda, Maryland.
Sirimontaporn, P. (1988). Introduction to the Taxonomy and Biology of the Seabass,
Lates calcarifer. In Seabass (Lates calcarifer) Culture in Thailand. FAO
Training Manual 88/3. Retrieved June 25, 2008, from
http://www.fao.org/docrep/field/003/ab707e/AB707E01.htm
44
Sriphairoj, K., Kamonrat, W., & Na-Nakorn, U. (2007). Genetic aspect in broodstock
management of the critically endangered Mekong giant catfish,
Pangasianodon gigas, in Thailand. Aquaculture, 264, 36-46.
Stuart, I.G., & McKillup, S.C. (2002). The use of sectioned otoliths to age barramundi
(Lates calcarifer) (Bloch, 1790) [Centropomidae]. Hydrobiologia, 479 (1-3).
231-236.
Terzioglu, M., & Larson. N. G. (2007). Mitochondrial Dysfunction in Mammalian
Ageing. In Mitochondrial biology: new perspectives. Wiley, Chichester
(Novartis Foundation Symposium 287), 197–213.
Thai, B.T., Pham, T.A. & Austin, C.M. (2006). Genetic diversity of common carp in
Vietnam using direct sequencing and SSCP analysis of the mitochondrial
DNA control region. Aquaculture, 258, 228-240.
Weiss, S., Schlotterer, C., Waidbacher, H., & Jungwirth, M. (2000). Haplotype
(mtDNA) diversity of brown trout Salmo trutta in tributaries of the Austrian
Danube: Massive introgression of Atlantic basin fish – by man or nature?.
Molecular Ecology, 10, 1241-1246.
Thai, B.T., Burridge, C.P. and C.M. Austin (2007). Genetic diversity of common carp
(Cyprinus carpio L.) in Vietnam using four microsatellite loci. Aquaculture,
269, 174-186.
Zhu, Z.Y. , Lin , G., Lo, L.C., Xu, Y.X., Feng, F., Chou, R. & Yue, G.H. (2006)
Genetic analyses of Asian seabass stocks using novel polymorphic
microsatellites. Aquaculture, 256, 197-173.