Terrestrial adaptation: Expansion of myoglobin and up-regulation of hemoglobin genes in the walking catfish (Clarias batrachus) by Ning Li A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 5, 2017 Keywords: Clarias batrachus, genome, adaptation, evolution, duplication, air-breathing organ Copyright 2017 by Ning Li Approved by Zhanjiang Liu, Chair, Professor of School of Fisheries, Aquaculture, and Aquatic Sciences Rex Dunham, Professor of School of Fisheries, Aquaculture, and Aquatic Sciences Charles Y. Chen, Professor of Crop, Soil and Environmental Sciences Aaron M. Rashotte, Associate Professor of Biological Sciences Abstract Walking catfish (Clarias batrachus), a freshwater fish capable of locomotion on land, serves as a great model for understanding adaptations to terrestrial life. Through comparative genome analysis, here I report that no genes were found within the walking catfish genome specifically related to the air-breathing capability. Instead, adaptive evolution was found in the C. batrachus genome to be involved in gene expression and nitrogenous waste metabolic process. Furthermore, myoglobin, olfactory receptor related to class A G protein-coupled receptor 1, and sulfotransferase 6b1 gene families were found specifically expanded in the air- breathing walking catfish genome with 15, 12, and 15 copies, respectively, while non-air- breathing fishes possess only 1-2 copies of these genes. Additionally, comparative transcriptome analysis between the air-breathing organ and the gill demonstrates the mechanism of aerial respiration involved in elastic fiber formation, oxygen binding and transport, and angiogenesis. Especially hemoglobin genes were expressed dramatically higher in the air-breathing organ than in the gill of C. batrachus. It is apparent that the coupling of enhanced abilities in oxygen transport and oxygen storage through genomic expansion of myoglobin and transcriptomic up- regulation of hemoglobin is an important component of the molecular basis for the adaptation of aquatic species to terrestrial life. ii Acknowledgements I would like to express my sincere gratitude to my major advisor. Dr. Zhanjiang Liu, for his constant guidance and support throughout my Ph.D. study. I am also grateful to my committee members: Dr. Rex Dunham, Dr. Charles Y. Chen and Dr. Aaron M. Rashotte and my university reader Dr. Joseph Kloepper for their assistance and time on my dissertation work. I would also like to thank Ludmilla Kaltenboeck for her technical assistance on my experiments. Many thanks also go to Dr. Shikai Liu, Dr. Jun Yao, Dr. Yun Li, Dr. Chen Jiang, Dr. Lisui Bao, Dr. Xiaoyan Xu, Dr. Yang Liu, Dr. Chenglong Wu, Dr. Sen Gao, Dr. Qifan Zeng, Dr. Xiaozhu Wang, Tao Zhou, Qiang Fu, Changxu Tian, Wendy Prabowo, Yulin Jin, Yujia Yang, Zihao Yuan, Suxu Tan, Huitong Shi, Wenwen Wang and all the other colleagues in the laboratory for their help, collaboration and friendship. I would like to thank Chinese Scholarship Council for the financial support during four years. Finally, I am grateful to my parents and my girlfriend Xinmi Zhang for their love and support. iii Table of Contents Abstract ............................................................................................................................................ ii Acknowledgements ......................................................................................................................... iii List of Tables .................................................................................................................................. vi List of Figures ................................................................................................................................ vii List of Abbreviations .................................................................................................................... viii LITERATURE REVIEW ............................................................................................................... 1 Air-breathing fishes and their air-breathing organs .................................................................... 1 The adaptive mechanisms corresponding to the bimodal respiration lifestyle ........................... 2 Walking catfish (Clarias batrachus) and its air breathing organ................................................ 6 Genetic and genomic studies of air-breathing fish species ......................................................... 8 INTRODUCTION ........................................................................................................................ 11 MATERIALS AND METHODS.................................................................................................. 13 C. batrachus samples and genome sequencing ......................................................................... 13 Genome assembly and assessment ............................................................................................ 13 Genome annotation ................................................................................................................... 14 Comparative genomic analysis.................................................................................................. 15 Gene family analysis ................................................................................................................. 18 Comparative transcriptomic analysis between gill and the air-breathing organ ....................... 19 Quantitative real-time PCR validation of differentially expressed genes ................................. 20 iv RESULTS AND DISCUSSION ................................................................................................... 22 Genome assembly and annotation ............................................................................................. 22 Comparative genomic analysis.................................................................................................. 26 Gene expansion ......................................................................................................................... 32 Expression of significantly expanded gene families ................................................................. 40 Comparative transcriptomic analysis between gill and the air-breathing organ ....................... 42 CONCLUSION ............................................................................................................................. 50 REFERENCES ............................................................................................................................. 52 APPENDICES .............................................................................................................................. 64 v List of Tables Table 1........................................................................................................................................... 20 Table 2........................................................................................................................................... 22 Table 3........................................................................................................................................... 23 Table 4........................................................................................................................................... 23 Table 5........................................................................................................................................... 24 Table 6........................................................................................................................................... 24 Table 7........................................................................................................................................... 28 Table 8........................................................................................................................................... 29 Table 9........................................................................................................................................... 31 Table 10......................................................................................................................................... 40 Table 11......................................................................................................................................... 42 Table 12......................................................................................................................................... 44 Table 13......................................................................................................................................... 46 Table 14......................................................................................................................................... 47 vi List of Figures Figure 1 ......................................................................................................................................... 25 Figure 2 ......................................................................................................................................... 26 Figure 3 ......................................................................................................................................... 27 Figure 4 ........................................................................................................................................