Title Page
Contents
List of Abbreviations 11
Abstract 13
Introduction 15
1. Background of the Study 15
2. Biology of African small barb Enteromius thysi 18
3. Taxonomy of E. thysi (Trewavas, 1974) 19
4. Mitochondrial DNA (mtDNA) in fishes 21
Materials and Methods 23
1. Sample Collection 23
2. gDNA Extraction 23
3. PCR amplification and partial sequencing for COI region 25
4. Primer walking 27
5. Sequence assembly and genome annotation 28
6. Data Analysis 30
Results and Discussion 32
1. Molecular Identification of Enteromius thysi 32
2. Genome structure, organization and composition 32
3. Protein-coding genes 33
4. Ribosomal RNA genes and transfer RNA genes 39
5. Genetic distances, phylogenetic relationship and biogeographic pattern 41
Conclusion 47
References 49
Appendices 57
Table 1. Primers used for the PCR-amplification of E. thysi mitogenome sequences 29
Table 2. Characteristic features of the mitochondrial genome of E. thysi. 34
Table 3. Nucleotide composition and skewness of Enteromius species mitogenomes. 36
Table 4. Nucleotide composition of PCGs of E. thysi mitochondrial genome. 40
Appendix Table 1. List of the species under with complete mitochondrial genome used in this study from NCBI website. 57
Appendix Table 2. List of designed primers used for long PCR of E. thysi 58
Appendix Table 3. Nested PCR condition amplifying COI-COIII contig of E. thysi 59
Appendix Table 4. Nested PCR condition amplifying COIII-NAD6 contig of E. thysi 60
Appendix Table 5. PCR condition amplifying NAD6-Dloop contig of E. thysi 60
Appendix Table 6. PCR condition amplifying D-loop-12S contig of E. thysi 61
Appendix Table 7. PCR condition amplifying 12S-16S contig of E. thysi 61
Appendix Table 8. Nested PCR condition amplifying 16S-COI contig of E. thysi 62
Appendix Table 9. Gene features of the Enteromius species and 2 Barbus outgroups 63
Fig 1. Map showing location for sampling site of E. thysi specimens in Nyong River, Akonolinga, Cameroon (3°45'53.8"N 12°14'48.5"E). 24
Fig 2. Mitochondrial genome of Enteromius thysi. tRNAs are encoded based on their abbreviations. GC content is plotted using black sliding window, GC-skew is plotted using... 38
Fig 3. The secondary structures of the 22 tRNA genes in the mitogenome of E. thysi. 42
Fig 4. K2P Genetic distances between E. thysi and other Enteromius species. 45
Fig 5. The Bayesian (BA) topology tree of E. thysi and other Enteromius species from Africa. The green color shows the Enteromius species distributed in the Northwest River... 46
Appendix Figure 1. Performing (left) genomic extraction inside the laminar flow hood and (right) visualization of the gel electrophoresis using UV light captured in digital camera. 75
Appendix Figure 2. Results of 1% agarose gel electrophoresis of the amplified PCR products targeting contig: (A) COI-COIII; (B) COIII-NAD6; (C) NAD6-... 76