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Copy of Molecular genetic studies on Tilapia fish

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mohamed adl

on 23 March 2015

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Transcript of Copy of Molecular genetic studies on Tilapia fish

A Thesis presented
By


Molecular Genetics Studies on Tilapia Fish

Aim of the work
Conclusions
1- Genetic Variation among tilapia fish species in Egypt using RAPD technique.
Material and Methods
Part one
Mayar Othman El_Sayed Ali
Under supervision of

Professor of Animal Husbandry
and Management,
Head of Animal Management and
Animal Wealth
Faculty of Veterinary Medicine,
Mansoura University

Prof. Dr.
Shabaan A. Hemeda

Professor of Genetics and Genetic Engineering,
Faculty of Veterinary Medicine,
Alexandria University

Dr. Salah M. Abdel-Rahman

Associate Professor of Molecular Genetics,

Nucleic Acid Research Department,
Genetic Engineering and Biotechnology,
Results
Part One
Introduction
Tilapia
has become increasingly important in aquaculture in tropical and subtropical countries. It is much appreciated by consumers, being a good and affordable source of protein.
Thesis entitled
In fishes,
RAPD fingerprinting
has been used for the detection of
DNA polymorphisms
in color mutant varieties of guppy, and differentiation of different species of fishes. Also it used to construct a genetic linkage map between different types of fish.
Components of the
growth hormone axis
plays a key role in regulating the growth process of fish; therefore, geneticists have sought to find alleles of genes in the growth hormone axis that are associated with low and high growth phenotypes.
The objectives of the present study were to reveal:
2- Growth hormone gene polymorphism in tilapia nilotica fishes and its association with growth rate by using two molecular genetic marker:
B- Single nucleotide polymorphism (SNP) using DNA sequencing technique.
A-Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).
The present work was carried out at the Nucleic Acids Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research & Technological Applications, Alexandria, Egypt.
Part two
Phylogenetic relationship between the three species of tilapia
Association between body weight and Polymorphism in growth hormone gene in tilapia nilotica
In the present study, three pure tilapia species (
Oreochromis niloticus, Oreochromis aureus, and Tilapia zillii
) were precisely selected from
Agricultural Research Center Abbassa, Abou Hammad, Governated Sharkia, Egypt
. However,
ten
fish from each species were used for this study.
3- RAPD – PCR
2- DNA Pooling
1-DNA isolation
Thermal cycling was carried out by
initial denaturation at 94 ºC for 4 minutes
, followed by
40-45 cycles
each at
94 ºC for 30-60 seconds
,
annealing temperature at 28-45 ºC for 30-60 seconds
and
polymerization temperature at 72 ºC for 1 min
.
Final extension was done at 72 ºC for 10 minutes
then the samples were held at 4 ºC.
5- Data analysis
(Statistica 5)
4- Electrophoresis
In the present study,
fifty tilapia nilotica
fish were precisely selected from a El-Manzala farm Dakahlia. The fishes selected from three grads of fish sizes:
large (weight ranged from 600.1gm to 356.5 gm)
,
medium (weight ranged from 240.5gm to 139.0 gm)
and
small (weight ranged from 062.0 gm to 019.0 gm)
.
1-DNA Extraction
2- PCR
PCR was done for amplification of the
2nd exon
,
2nd intron
,
3rd exon
,
3rd intron
and
4th exon
of the
growth hormone gene (GH)
with expected amplicon size of
932 bp
.
3-Restriction Fragment Length Polymorphism (RFLP)
The amplified DNA fragment of the growth hormone gene was digested with
restriction enzymes.
4- Electrophoresis
5- DNA sequencing
6-Data analysis
•Calculation of gene and genotypic frequencies
•Determination of association between fish size and GH polymorphism (genotypes) by using SAS, (2004)
City of Scientific Research and Technology Applications, Alexandria
Part Two
Primer 5
Primer 10

This parsimony tree divides the three fish species into
two different clusters
.
Cluster I
contains the two species belong to the genus Oreochromis (O. aureus and O. niloticus), while
cluster II
tilapia zillii fish species. contains
RAPD analysis
Parsimony tree
Primer 2
Primer 7
PCR product of GH (965 bp)
RFLP
Restriction analysis of PCR-RFLP-of GH gene were performed by using 10 RE (
Eco RI – Eco RV – Bam HI- Hind III- Ava II – ApaI – BIPI- HincII – AluI – sacI - TaqI
), some of them digested the amplified fragment (
Alu 1- HincII - SacI – Taq I
) showing no differences between the different fish under the study. The other restriction enzymes did not digest this fragment in all fish.
HINFI
was the only RE that digested the 965-bp fragment and showed differences between the different fish under the study.
Genotypes of large-sized fish
Genotypes of small-sized fish
Gene and genotype frequencies
LSM ± SE of body weight and genotypes of tilapia nilotica fish
Phylogenetic tree of GH gene with other four GH genes
DNA Sequencing
1- The three famous types of tilapia fish in Egypt can be classified into two clusters
Cluster I
contain the two species belongs to the genus Oreochromis, while
cluster II
contains tilapia zillii fish species.
2- Using
PCR-RFLP
method, to study the association between growth hormone gene genotypes and growth rate in tilapia fish, there was no significant association between body size trait and growth hormone genotype.
3-Using
SNPs
in growth hormone gene in selected tilapia niloticus fish can be used for marker assisted selection (MAS) as part of nucleotide sequence of fish. and It can be used for marker assisted selection for large size fish to increase and
accelerate the rate of genetic improvement on

white meat production trait
.
Primer 1
Primer 3
Primer 4
Primer 6
Primer 9
Primer 8
Primer 11
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Professor of Animal Husbandry
and Management,
Head of Animal Management and
Animal Wealth
Faculty of Veterinary Medicine,
Mansoura University
Full transcript