Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish by Ruijia Wang 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 December 14, 2013 Key word: Bulk segregant analysis (BSA); Bulk segregant RNA-seq (BSR-Seq); Bulk frequency ratio (BFR); Disease resistance; catfish; Allele-specific expression Copyright 2013 by Ruijia Wang Approved by Zhanjiang Liu, Chair, Alumni Professor, School of Fisheries David Rouse, Alumni Professor, School of Fisheries, Nannan Liu, Professor, Department of Entomology and Plant Pathology Jeffery Terhune, Associate Professor, School of Fisheries Abstract The application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the correlated phenotypes. In this study, we demonstrated the use of bulked segregant RNA-seq (BSR-Seq) for the analysis of differentially expressed genes and associated SNPs with disease resistance against enteric septicemia of catfish (ESC). A total of 1,255 differentially expressed genes were found between resistant and susceptible fish. In addition, 56,419 SNPs were identified as significant SNPs between susceptible and resistant fish located on 4,304 unique genes. Mapping of the significant SNPs, along with analysis of differentially expressed genes, allowed identification of candidate genes underlining disease resistance against ESC disease. This study demonstrated the use of BSR-Seq for the identification of genes involved in disease resistance against ESC through expression profiling and mapping of significantly associated SNPs. BSR-Seq is applicable to analysis of genes underlining various performance and production traits without significant investment in the development of large genotyping platforms such as SNP arrays. ii Acknowledgments The author is very grateful to Dr. Zhanjiang Liu for his constant guidance, support and patience in the past years. I have been very fortunate in having Dr. Zhanjiang Liu as my supervisors. His scientific attitude and philosophy have had a profound influence on me. I am also is grateful for time and expertise offered to me by my committee members: Dr. Nannan Liu, Dr. David Rouse and Dr. Jeffery Terhune, and the dissertation outside reader, Dr. Bernhard Kaltenboeck. I would also like to express my gratitude to Huseyin Kucuktas and Ludmilla Kaltenboeck for technical help, and the colleagues in the laboratory for their help, collaboration, and friendship. I am grateful to my parents and my beloved wife for their endless love and support. iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments .......................................................................................................................... iii List of Tables .................................................................................................................................. ii List of Illustrations ......................................................................................................................... iii List of Abbreviations ..................................................................................................................... iv I. Introduction and literature review ............................................................................................... 1 I.1 ESC disease in channel catfish .......................................................................................... 1 I.2 SNPs studies in catfish and other species .......................................................................... 2 I.3 Deep sequencing of transcriptome response to bacterium infection ................................. 3 I.4 BSA and BSR-seq ............................................................................................................. 4 II Materials and methods................................................................................................................. 7 II.1 ESC bacterial challenge.................................................................................................... 7 II.2 Sampling and RNA isolation ............................................................................................ 8 II.3 Illumina sequencing ......................................................................................................... 8 II.4 De novo assembly ............................................................................................................. 9 II.5 Gene identification and annotation................................................................................... 9 II.6 Transcript-level gene expression analysis of different groups ....................................... 10 II.7 Sequencing mapping and significant SNP identification ............................................... 10 II.8 Analysis of bulk frequency ratios and allele specific expression ................................... 11 ii II.9 Genomic location of ESC resistance-related genes ........................................................ 12 III Results ...................................................................................................................................... 12 III.1 Sequence assembly and analysis ................................................................................... 12 III.2 Differentially expressed genes after infection .............................................................. 14 III.3 Comparison of gene expression in resistant fish and susceptible fish after infection ................................................................................................................................ 15 III.4 Identification of SNPs and significant SNPs ................................................................ 51 III.5 Bulk frequency ratios .................................................................................................... 52 III.6 Genes with large BFR caused by genetic segregation .................................................. 63 III.7 Genes with large BFR caused by allele-specific expression......................................... 67 III.8 Location of genes with high bulk frequency ratio (BFR) ............................................. 70 III.9 Parental origin of highly expressed alleles ................................................................... 76 IV Discussion ................................................................................................................................ 79 Involves in polyamine homeostasis .............................................................................................. 86 V Conclusions ............................................................................................................................... 89 Reference ...................................................................................................................................... 90 iii List of Tables Table 1 ....................................................................................................................................... 20 Table 2 ....................................................................................................................................... 21 Table 3 ....................................................................................................................................... 23 Table 4 ....................................................................................................................................... 24 Table 5 ....................................................................................................................................... 56 Table 6 ....................................................................................................................................... 59 Table 7 ....................................................................................................................................... 60 Table 8 ....................................................................................................................................... 73 Table 9 ....................................................................................................................................... 74 Table 10 ..................................................................................................................................... 92 ii List of Figures Figure 1 ...................................................................................................................................... 58 Figure 2 ...................................................................................................................................... 71 Figure 3 ...................................................................................................................................... 72 Figure 4 ...................................................................................................................................... 79 Figure 5 .....................................................................................................................................