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Copy of Copy of Molecular genetic studies on Tilapia fish

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Mohammed Rizk

on 5 February 2013

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Transcript of Copy 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 Prof. Dr.
Mohamed M. Fouda
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 jhhhghlhgf
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