Title Page
Contents
Chapter 1. The species identification based on whole mitogenome 11
1-1. Introduction 12
1-2. Materials and Methods 25
1-2-1. Sample 25
1-2-2. gDNA extraction and PCR 25
1-2-3. NGS 25
1-2-4. Annotation 27
1-2-5. Phylogenetic tree 27
1-3. Results 29
1-3-1. Relationship between total DNA quality and sequencing result by NGS 29
1-3-2. Identification of species 32
1-3-3. Annotation 34
1-3-4. Phylogenetic tree 48
1-4. Discussion 68
Chapter 2. Studies on the marker-assisted selection in marine organism: Utilization of single nucleotide polymorphisms in growth hormone gene for marker-assisted selection Nile tilapia (Oreochromis niloticus) 72
2-1. Introduction 73
2-2. Materials and Methods 77
2-2-1. Sample 77
2-2-2. Genomic DNA (gDNA) extraction 78
2-2-3. Sanger sequencing 78
2-2-4. Analysis for identifying the relationship between SNPs and body weight 81
2-2-5. Genotyping 82
2-2-6. Verification of primer sets and probes 85
2-2-7. Verification of the effects of GBs on the body weight through breeding 86
2-3. Results 87
2-3-1. Identification of SNPs 87
2-3-2. Relationship between SNPs and body weight 92
2-3-3. Relationship between GBs and weight 95
2-3-4. Genotyping 99
2-3-5. Genotyping accuracy 106
2-3-6. Breeding 108
2-4. Discussion 111
Reference 117
요약 135
Table 1-1. Comparison between NGS platforms. 21
Table 1-2. Studies that report the distinctive structures of tRNAs and D-loop in metazoan mitogenomes. 23
Table 1-3. Studies that report the mitogenome structures. 24
Table 1-4. Information of NGS raw data from total DNA. 31
Table 2-1. Primer sets used in this study. 80
Table 2-2. Oligo information for genotyping of growth hormone in Nile tilapia. 84
Table 2-3. Individual number and average body weight according to alleles in each SNP observed in Nile tilapia male and female. 90
Table 2-4. Genotypic blocks observed in Nile tilapia. 91
Table 2-5. Accuracy of qPCR kit for genotyping of growth hormone in Nile tilapia. 107
Table 2-6. Proportion of individuals that retain good genotypic block in parents and F1 fry. 110
Figure 1-1. Three types of total DNA quality used in this study. 30
Figure 1-2. The complete mitochondrial genome map of Notostomum cyclostomum. 40
Figure 1-3. Gene order comparison the mitogenome from Syllis sp. and closely related species. 41
Figure 1-4. The complete mitochondrial genome map of Pseudopleuronectes herzensteini. 42
Figure 1-5. The complete mitochondrial genome map of Microstomus achne. 43
Figure 1-6. The complete mitochondrial genome map of Acanthopsetta nadeshnyi. 44
Figure 1-7. The complete mitochondrial genome map of Dexistes rikuzenius. 45
Figure 1-8. The complete mitochondrial genome map of Hippoglossoides dubius. 46
Figure 1-9. The complete mitochondrial genome map of Laeops kitaharae. 47
Figure 1-10. Phylogenetic tree of Notostomum cyclostomum and related species using cox1. 50
Figure 1-11. Phylogenetic tree of Notostomum cyclostomum and related species using core mitogenome. 51
Figure 1-12. Phylogenetic tree of Notostomum cyclostomum and related species using cox1. 52
Figure 1-13. Phylogenetic tree of Syllis sp. and other species based on core mitogenome. 53
Figure 1-14. Phylogenetic tree of 18S rRNA sequences from Syllis sp. and other species of the family Syllidae. 54
Figure 1-15. Phylogenetic tree of Pseudopleuronectes herzensteini and related species using cox1. 56
Figure 1-16. Phylogenetic tree of Pseudopleuronectes herzensteini and related species using core mitogenome. 57
Figure 1-17. Phylogenetic tree of Microstomus achne and related species using cox1. 58
Figure 1-18. Phylogenetic tree of Microstomus achne and related species using core mitogenome. 59
Figure 1-19. Phylogenetic tree of Acanthopsetta nadeshnyi and related species using cox1. 60
Figure 1-20. Phylogenetic tree of Acanthopsetta nadeshnyi and related species using core mitogenome. 61
Figure 1-21. Phylogenetic tree of related flatfish species including Rikuzen flounder (Dexistes rikuzenius) using cox1. 62
Figure 1-22. Phylogenetic tree of related flatfish species including Rikuzen flounder (Dexistes rikuzenius) using core mitogenome. 63
Figure 1-23. Phylogenetic tree of Hippoglossoides dubius and related species using cox1. 64
Figure 1-24. Phylogenetic tree of Hippoglossoides dubius and related species using core mitogenome. 65
Figure 1-25. Phylogenetic tree of Laeops kitaharae and related species using cox1. 66
Figure 1-26. Phylogenetic tree of Laeops kitaharae and related species using core mitogenome. 67
Figure 2-1. Gene structure of growth hormone (GH) in Nile tilapia and single nucleotide polymorphisms (SNPs) discovered in this study. 89
Figure 2-2. Relationship between alleles of each SNP and body weight of Nile tilapia male. 93
Figure 2-3. Relationship between alleles of each SNP and body weight of Nile tilapia female. 94
Figure 2-4. Relationship between genotypic blocks (GBs) and body weight of Nile tilapia. 97
Figure 2-5. PCA for identifying valuable genotypic blocks (GBs) in growth hormone (GH) of Nile tilapia. 98
Figure 2-6. Minimum target single nucleotide polymorphism (SNPs) for determination of each genotypic blocks (GBs). 101
Figure 2-7. Detection of allele of 3US1 in OnGH 3'-UTR using PNA probe. 102
Figure 2-8. Detection of allele of 3US1 in OnGH 3'-UTR using DNA probe. 103
Figure 2-9. Detection of allele of PS3 in OnGH promoter using DNA probe. 104
Figure 2-10. Detection of allele of FIS3 in OnGH ORF using DNA probe. 105
Figure 2-11. Growth curve of Nile tilapia F1 fry. 109