Title Page
요약
Abstract
Contents
List of Abbreviations 15
Chapter 1. General Introduction 17
1.1. Introduction 18
1.1.1. Historical Background of Fisheries and Aquaculture 18
1.1.2. Fish as a Source of Protein 19
1.1.3. Aquaculture 19
1.2. Nile tilapia (Oreochromis niloticus): Biology and Habitat 20
1.2.1. Genetic Characterization of Nile Tilapia 22
1.2.2. Single Nucleotide Polymorphism (Types and Importance) 23
1.3. Muscle Hormone Genes: Origin, Development, and Growth 25
1.3.1. Myogenin Gene in the Nile Tilapia/Aquaculture 26
1.3.2. Myostatin Gene in the Nile Tilapia/Aquaculture 26
1.4. Quantitative Polymerase Chain Reaction 28
1.5. Single Nucleotide Polymorphism and Quantitative PCR 29
1.6. Objectives of the study 30
Chapter 2. SNP Discovery in Muscle Hormone Genes and Their Association with Body Weight in the Nile Tilapia 32
2.1. Introduction 33
2.2. Materials and Methods 34
2.2.1. Ethical statement 34
2.2.2. Tilapia Farming for SNP Analysis 34
2.2.3. Experimental Design and Collection of Samples 36
2.2.4. Extraction of DNA and Amplification of PCR 36
2.2.5. Sequence Analysis, and Discovery of SNP 39
2.3. Results 40
2.3.1. SNP Discovery 40
2.3.2. Association between SNP and Body weight 45
2.4. Discussion 47
2.4.1. Discovery of SNP 47
2.4.2. Association between SNP and Body weight 49
Chapter 3. Relationship between Body Weight and Genotype Block in the Nile Tilapia 51
3.1. Introduction 52
3.2. Materials and Methods 54
3.2.1. Statistical Analysis 54
3.2.2. Principal Component Analysis 54
3.3. Results 55
3.3.1. Association between Body weight and Genotype Block 55
3.4. Discussion 69
3.4.1. Association between Body Weight and Genotype Block 69
Chapter 4. Designing of Genetic Marker Kit using Quantitative PCR 71
4.1. Introduction 72
4.1.1. Quantitative PCR Application in Aquaculture 73
4.1.2. Genetic Markers in Aquaculture 74
4.1.3. Types of Genetic Markers and their Application 75
4.2. Amplification Curve Analysis 77
4.2.1. Ct Curve Analysis 78
4.2.2. Melting Temperature Analysis 79
4.3. Materials and Methods 80
4.3.1. Primer and Probe Designing for Quantitative PCR 80
4.3.2. DNA Isolation and Amplification of Quantitative PCR 84
4.4. Results 91
4.4.1. Genotyping by Ct Value and Melting Temperature Curve Analysis 91
4.4.2. Screening of Ct value and Melting Temperature Curve 93
4.4.3. Detection Kit 103
4.4.4. Growth of F1 Generation 109
4.5. Discussion 112
Chapter 5. General Discussion 115
5.1. General Discussion 116
5.2. Concluding Remark 123
Future Directions 124
References 126
Publications 150
Table 1. Primer sets used for amplification of MyoG and MSTN genes 38
Table 2. SNPs/Insertion in MyoG gene 43
Table 3. SNPs/Insertion in MSTN gene 44
Table 4. Genotype block composition of MyoG gene 58
Table 5. Genotype block composition of MSTN gene 61
Table 6. Primer and Probe set used for qPCR amplification of MyoG gene 82
Table 7. Primer and Probe set used for qPCR amplification of MSTN gene 83
Table 8. Genetic marker screening kit analysis for MyoG gene 94
Table 9. Genetic marker screening kit analysis for MSTN gene 96
Figure 1. Identification of SNPs in the Oreochromis niloticus OnMyoG gene. 41
Figure 2. Identification of SNPs in the Oreochromis niloticus OnMSTN gene. 42
Figure 3. Descriptive statistical analysis of the weight by a genotype block of OnMyoG male. 56
Figure 4. Descriptive statistical analysis of the weight by a genotypic block of OnMyoG female. 57
Figure 5. Descriptive statistical analysis of the weight by a genotypic block of OnMSTN male. 59
Figure 6. Descriptive statistical analysis of the weight by a genotypic block of OnMSTN female. 60
Figure 7. Scatter plot of principal component axis one dimension 1 (Dim1) and axis two dimension 2 (Dim2) based on SNP data of OnMyoG male. 65
Figure 8. Scatter plot of principal component axis one dimension 1 (Dim1) and axis two dimension 2 (Dim2) based on SNP data of OnMyoG female. 66
Figure 9. Scatter plot of principal component axis one dimension 1 (Dim1) and axis two dimension 2 (Dim2) based on SNP data of OnMSTN male. 67
Figure 10. Scatter plot of principal component axis one dimension 1 (Dim1) and axis two dimension 2 (Dim2) based on SNP data of OnMSTN female. 68
Figure 11. qPCR conditions used for tilapia MyoG SNP 3'US1 site genotyping 85
Figure 12. qPCR conditions used for tilapia MyoG SNP 3'US2 site genotyping 86
Figure 13. qPCR conditions used for tilapia MyoG SNP 3'US3 site genotyping 87
Figure 14. qPCR conditions used for tilapia MSTN SNP 5'US2 site genotyping 88
Figure 15. qPCR conditions used for tilapia MSTN SNP 3'US1 site genotyping 89
Figure 16. qPCR conditions used for tilapia MSTN SNP 3'US2 site genotyping 90
Figure 17. Melting temperature cure detection of MyoG-3US1. 97
Figure 18. Melting temperature cure detection of MyoG-3US2. 98
Figure 19. Ct value detection of MyoG-3US3. 99
Figure 20. Ct value detection of MSTN-5US2. 100
Figure 21. Ct value detection of MSTN-3US1. 101
Figure 22. Ct value detection of MSTN-3US2. 102
Figure 23. Tilapia Genetic Marker Detection Kit Prototype 107
Figure 24. Components of Prototype Kit for Detecting Tilapia Gene Marker 108
Figure 25. The growth curve of tilapia fry is shown in this figure, with varying growth rates denoted by distinct colored lines. 111