MITOCHONDRIAL DNA VARIATION OF CULTURED

AND WILD ASIAN SEABASS (Lates calcarifer) IN

THAILAND

Yusmansy a
ID.
h
50912419

Department of Aquatic Science, Faculty of Science,

Burapha University
I. Introduction
Statement and Significance of The Problem
Objectives
Contribution to Knowledge
Scope of Study
Hypothesis

II. Literature Review

Biology of Asian Seabass

Ecology and distribution

Genetic Processes in Populations

mtDNA as genetic marker for population genetic analyses

Molecular techniques for mtDNA analysis

 I.
•Statement and Significance of The Problem

Introduction
•Objectives
•Contribution to Knowledge
•Scope of Study
•Hypothesis
Background
I.
Introduction

FAO, 2006
Background
I.
Introduction
• Thailand is a major producer of Asian seabass
(Lates calcarifer) fingerlings, mainly produced in
hatcheries

• Well managed breeding program is

important to produce good quality
fingerlings

• Success of a breeding program highly

depends on broodstock management
maintaining genetic diversity
 I.
Problem
Introduction
Loss genetic diversity in hatcheries due to inapropriate
hatchery practises, such as:

- Mating limited number of broodstock

- Mass spawning that lead to unequal sex
ratio and contribution of each families
 I.
Consequences of the Problem
Introduction
Reduced genetic diversity may lead to undesirable
consequences on traits reated to production, for
instance:

- Resistance to the disease stress

- Adaptive potential to the environmental
stress
How to overcome the Problem
Managing broodstock that able to maintain genetic
diversity.
Broodstock collection
Design of mating scheme
Rearing practice

Introducing variation from Wild populations.

Wild populations are potential source of genetic
variation, because wild populations usually have larger
population size (Ne) -> likely contain higher genetic
variation

To implement both alternatives, genetic data

Molecular tools to evaluate
genetic data
Heterozygosity of nuclear DNA and genetic distance
Allozyme
Sequencing
Microsatellite

Haplotype and nucleotide diversity of mtDNA

Genomic RFLP
PCR -RFLP
Direct sequencing
Molecular tools to evaluate
genetic data (cont.)
Smaller genome size than nuclear DNA

mtDNA is generally very sensitive to detect

population structure due to lower Ne than
nuclear DNA

Nucleotide substitution rate higher than

nuclear DNA

Not subject to recombination

Objective of the Study
1 Estimate genetic variation within hatchery, and wild
populations of L. calcarifer using PCR-RFLP of the D-
loop control region of mitochondrial DNA

2 Examine population differentiation among hatchery

and wild populations
Contribution to knowledge
1 The genetic data will be useful in managing existing genetic diversity in hatchery
populations of L. calcarifer in Thailand

2 This data should be useful for breeders and government in order to develop a selective
breeding program for L. calcarifer species

3 Data for wild populations will provide basic information to aid conservation efforts of
native genepool of L. calcarifer in Thailand
Scope of study
1 Analysis: mtDNA control region
2 Samples taken from 3 hatchery and 2 wild populations located around Gulf of Thailand,
represent broodstock supplying fingerling in Thailand

3 Technique: Polymerase Chain Reaction (PCR) – Restriction Fragment Length

Polymorphisms (RFLP)
Hypothesis
1 Level of genetic variation within hatchery populations of L. calcarifer is lower than of wild
populations.

2 Genetic differentiation among wild L. calcarifer populations is high, but the differences
among hatchery populations are low
II. Literature
Review
• Biology of Asian Seabass
• Ecology and distribution
• Genetic Processes in Populations
• mtDNA as genetic marker for population genetic
analyses
• Molecular techniques for mtDNA analysis
• Genetic Diversity of Asian seabass and fish
species with similar life history
 Literat Biology of
Rure
eview
Asian Seabass

Classification
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Perciformes
Family: Centropomidae
Genus: Lates
Species: Lates calcarifer
Source: fishase.org
 Literat Biology of
Rure
eview
Lates calcarifer

Catadromous-demersal (Moore, 1982)

Larvae and young juveniles live in estuaries,

older juveniles inhabit the upper reaches of
rivers (Moore & Reynolds, 1982)

Vertical migration reaches maximum 70 Km,

horizontal migration maximum 17 Km (Russel &
Garrett, 1988)
 Literat Biology of
Rure
eview
Lates calcarifer
Growth Parameter:

Protandrous hermaphrodites
Individual is sexualy matured as male in the first time, then
gonad structure develops into female in the following ages
(Moore, 1979)
 Literat Ecology &
Rure
eview
Distribution

Single annual reproductive period (Davis, 1985;

Guiguen et al., 1994)

Adult -> Carnivorous;

Juveniles -> Omnivorous (Sirimontaporn, 1988)

Important food: Crustacean, decapoda,

mysidacea, Isopoda (Cabral & Costa, 2001), and
Amphipods (Laffaile et al., 2001)
 Literat Ecology &
Rure
eview
Distribution

Asian seabass distributed

spread along Indo-West
Pacific: eastern edge of the
Persian Gulf to China,
Taiwan and southern Japan,
southward to southern Papua
New Guinea (Larson, 1999),
Indonesia and Australia
(Marshall, 2005)

World geographic distribution of L.

calcariifer (Retrieved from FIGIS-FAO,
http://www.fao.ofg/figis/)
 Literat Genetic Processes
Rure
eview
In Populations

Mutations
Primary source of genetic variation in
populations

Two major types of mutations: Point (gene) mutations and

Chromosomal mutations

Change could be due to base pair substitution, insertion or deletion

Chromosomal mutation
 Literat Genetic Processes
Rure
eview
In Populations

Genetic drift
Is change in allele frequency in a population in
succesive generations due to random process

Magnitude of genetic drift in a population depend on deviation level from an ideal

condition

In general, genetic drift occurs when the founder population size is small

Mitochondrial DNA has lower population size (Ne) than nuclear DNA, therefore
more susceptible to genetic drift effects
 Literat Genetic Processes
Rure
eview
In Populations

Gene flow
Is any movement of alleles from one population to
another through recombination of sexual reproduction

Gene flow will increase or maintain genetic variation in a population, but

decrease distinctiveness among populations.

Gene flow in mtDNA can be indicated by haplotype share between genetically

related populations
 Literat Genetic Processes
Rure
eview
In Populations

Natural selection
Unequal probability of survived or reproduced alleles to
the future generation causes genetic variation change

Major factor leads to the natural selection process in a

populations the differential survival or reproduction
of genotypes

Alleles associated with favorable heritable traits

become more common, while unfavorable ones
become less common.
 Literat
Rure
eview
Mitochondrial DNA as genetic marker
for Population genetic Analysis

• Uniparental inheritance
• Relatively small effective population
size than nucleus DNA
• Higher mutation rate
• Powerful for detecting patterns of
genetic structure in natural systems

courses.washington.edu/fish340/Lab%20Pro
ject.htm
 Literat Molecular Techniques for
Rure
eview Mitochondrial DNA Analysis

Restriction Fragment Length Polymorphisms (RFLP)

Detecting nucleotide variation based on specific restriction site
recognition of :
• Whole genomic DNA, or
• Specific region, e.g. control region, cytochrome b

Direct Sequencing
Determination of the order of nucleotide bases, i.e. Adenine(A),
Guanine (G), Cytosine (C) and Thymine (T).
Direct sequencing has higher sensitivity in detecting nucleotide
Bernatchez & Danzmann (1993) suggested congruence of
the magnitude of variation and differentiation resulted from
RFLP and sequencing analysis
 Literat Genetic diversity of L. calcarifer
Rure
eview And fish species with similar life history

Genetic variation within population

• Genetic variation in wild population of L. calcarifer
is relatively high
 Literat Genetic diversity of L. calcarifer
Rure
eview And fish species with similar life history

Genetic variation within population

• Genetic variation in hatchery population is lower
than wild populations

• In some cases, genetic variation within hatchery

may higher than wild because of it consist of
individual coming from genetically distinct sources
 Literat Genetic diversity of L. calcarifer
Rure
eview And fish species with similar life history

Genetic differentiation among

populations
• Genetic differentiation among wild population is
much lower than within population, suggesting
presence of population structure
 Literat Genetic diversity of L. calcarifer
Rure
eview And fish species with similar life history

Migration pattern
• Indicated stepping stone model of migration one
dimensional linear structure along the river

• This migration pattern will preserve the genetic

diversity rather among population

Picture reference: Neal, D. (2004) Introduction to population biology. Cambridge University Press. Cambridge - UK
 Literat Genetic data
Rure
eview
Analysis
Genetic diversity within population
Haplotype diversity
Nucleotide diversity

Genetic differentiation among population

Mean number of genetic differentiation between two
randomly chosen haplotypes

Genetic differentiation also can be indicated by Φ-

statistics by using Analysis of Molecular Variation (AMOVA)
developed by Excoffier, Smouse & Quattro (1992)
 Literat Genetic data
Rure
eview
Analysis
Genetic distance
Genetic distance can be estimated from 3 ways:

• From the proportion of nucleotide substitution

• Pairwise FST values derived based on drift model
• Average number of restriction site differences between
populations (D)

Relationship among populations

Relationship among population can be constructed using
Neighbor-Joining (NJ) method based on pairwise genetic
distance values.
 Research
methodology

III. Research
Methodology
Samples collection
DNA extraction
PCR amplification
Enzyme digestion
Data Analysis
 Research Collecti
Sampl
es
methodology
on

3
2 1

4 5
 Research Collecti
Sampl
es
methodology
on
Sampling Design

30-40 individuals per sample

 Research Collecti
Sampl
es
methodology
on
• Cut small amount caudal fin
• Sample preserved in 95% Ethanol
• Labeling

Digestio
Cell
• Cut small amount 0,5 mm sample.
• Insert to Lysis buffer + Proteinase K
• Incubate overnight at 55oC
n
Research Extraction
DNA

methodology
Salting out method NaCl saturated H2O
 Research Amplification
PCR

methodology

PCR-Reaction • Primers – 2 pmole each

LN20 (5’-ACC ACT AGC ACC CAA AGC TA-3’)
HN20 (5’-GTG TTA TGC TTT AGT TAA GC-3’)
• 1x PCR Buffer
• 2mM dNTPs,
• 0.4 mM MgCl2,
• Taq Polymerase,
• dH2O
• DNA template

PCR-Profile
Initial denaturing 94oC 9
o
35
Cycles
4 7 Final extention 72oC 10
1 min 2 C 0oC 2oC min
60s 60s 90s
Research Digestion
enzym
e
methodology

7 Restriction enzymes will be used:

• HindII, FauI, EcoRV, EcoRI, BstENI, Bse1I, EcoNI

10 µl RFLP-Reaction contains:
• 1 µl - 1x Digestion Buffer
• 2 µl - PCR product template
• 0.1 µl – 2 u/ µl restriction enzyme
• 6.9 µl dH2O

Incubate overnight at 37oC (FauI); 37oC (HindII, EcoRI,

EcoRV, EcoNI); and 65oC(Bse1I & BstENI)
Research Digestion
enzym
e
methodology
Scoring design for composite haplotype assignment

Composite haplotype is then rearranged to assigned

the haplotype frequency
Research Digestion
enzym
e
methodology
Scoring for present/absence of the fragment position

The composite of present/absence (0/1 binary data

format) is required for data analysis in Arlequin
program, for example
RA1 01100011
RA2 10001000
RA3 01100110
Research Analysis data

methodology
Genetic diversity within populations
haplotype diversity within and population, derived from formula

where xi2 = frequency of sample of the ith haplotype; n = number of sample, l=number of nucleotide (Nei and Tajima, 1980)

Nucleotide diversity derived from formula proposed by Nei and Li (1990)

where xi and xj = frequency of ith and jth haplotype; Πij = number of nucleotide differences per nucleotide sites between ith and jth haplotype.
Π = Σij xi xj Πij
Haplotype and nucleotide diversity will be performed
using Analysis of Molecular variation (AMOVA) in the
Arlequin program
Research Analysis
data

methodology
Genetic differentiation among populations

Genetic differentiation 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.

ΦST ΦSC ΦCT

Where , , and are the associated covariance

component for groups, populations within a group, within
populations, and total variation.

These calculations can be performed by AMOVA

in the Arlequin program
Research Analysis
data

methodology
Genetic differentiation among populations

Design for hierarchical Analysis of Molecular Variance

(AMOVA)
Research Analysis
data

methodology
Genetic differentiation among populations

Design for hierarchical Analysis of Molecular Variance

(AMOVA) in the Arlequin program
Research Analysis
data

methodology
Genetic distance
Genetic distance can be calculated based on average number
of restriction site differences between populations (D) (Nei & Li,
1979). The difference between populations A and B
( AB)

where
k & k’ = 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

Distance between population 1 and 2, and other OTU j can be

calculated by

where D1j and D2j are the distance between OTU 1 and OTU j; and OTU 2 -
OTU j, respectively.
Research Analysis
data

methodology

Population relationships will be constructed using

Neighbor-Joining method based on Nei genetic
distance and visualized by a consensus (bootstrap
1000 replicates) using PHYLIP program

Consensed dendogram then will be visualized using

TreeView Program
 Thank
Thank You
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