Multi-Omics Analysis of a Rat Model of Aerobic Exercise Capacity and Metabolic Fitness by ...

150 Pages·2015·5.35 MB·English

Multi-Omics Analysis of a Rat Model of Aerobic Exercise Capacity and Metabolic
Fitness
by
Yangsu Ren

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Human Genetics)
in The University of Michigan
2016

Doctoral Committee:

Associate Professor Jun Li, Chair
Professor Steven L. Britton
Professor David T. Burke
Assistant Professor Jeffery M. Kidd
Professor Kerby A. Shedden
© Yangsu Ren 2016
DEDICATION

To my family and friends

ii
ACKNOWLEDGEMENTS

First, I have to thank my mother for her unwavering support. I also have to
acklowledge all of the friends I have made here at the University of Michigan, who have
all been great sources of inspiration.

I also have to thank my advisor, Jun Li, who is the most important figure of my
PhD. I cannot begin to express my appreciation for his support and dedication to my
growth as an independent scientist. I must also thank the past and present members of
the Li lab for their help and support.

Finally, I have to thank the members of my thesis committee, Dave Burke, Jeff
Kidd, Steve Britton and Kerby Shedden for providing me with guidance and expertise on
my dissertation project.

iii
TABLE OF CONTENTS
DEDICATION .................................................................................................................. ii
ACKNOWLEDGEMENTS .............................................................................................. iii
LIST OF TABLES ......................................................................................................... vii
LIST OF FIGURES ....................................................................................................... viii
LIST OF ABBREVIATIONS ............................................................................................ x
ABSTRACT ................................................................................................................... xi
CHAPTER 1: Introduction ............................................................................................. 1
1.1 Importance of rat models in studying complex traits ....................................... 1
1.2 Artificial selection for high and low aerobic capacity rats ................................ 2
1.3 Phenotypic Divergence Between HCR and LCR ............................................ 3
1.4 My contribution, using the HCR-LCR model for gene mapping ...................... 4
CHAPTER 2: Genetic Analysis of the Rat Pedigrees ................................................. 7
2.1 Introduction ..................................................................................................... 7
2.2 Materials and Methods ................................................................................... 7
2.2.1 Ethics Statement ............................................................................... 7
2.2.2 Rotational Breeding Scheme ............................................................ 8
2.2.3 Running Phenotype .......................................................................... 9
2.2.4 Phenotype Distribution .................................................................... 10
2.2.5 Inbreeding Coefficient and Heritability ............................................ 10
2.2.6 Genotyping and Data Processing ................................................... 12
2.2.7 Runs of Homozygosity .................................................................... 13
2.2.8 Genomewide Average Heterozygosity ............................................ 13
2.2.9 LD calculation ................................................................................. 13
2.3 Results.......................................................................................................... 14
2.3.1 Rotational Breeding and Inbreeding Coefficients ............................ 14
2.3.2 Phenotypic Response to Selection and Heritability ......................... 14
2.3.3 Increased Genomic Differentiation Between Lines ......................... 16
2.3.4 Decreased Genomic Diversity Within Lines .................................... 17
2.3.5 Linkage Disequilibrium .................................................................... 18
2.4 Discussion ................................................................................................... 18
iv
2.5 Figures.......................................................................................................... 24
2.6 Tables ........................................................................................................... 30
CHAPTER 3: Selection-, Age-, and Exercise-Dependence of Skeletal Muscle Gene
Expression Patterns .................................................................................................... 33
3.1 Introduction ................................................................................................... 33
3.2 Materials and Methods ................................................................................. 34
3.2.1 Ethics Statement ............................................................................. 34
3.2.2 Animals ........................................................................................... 34
3.2.3 Tissue and RNA extraction ............................................................. 35
3.2.4 Gene expression microarray ........................................................... 35
3.2.5 Gene expression data analysis ....................................................... 35
3.2.6 Pathway analysis ............................................................................ 37
3.3 Results.......................................................................................................... 37
3.3.1 Global patterns ............................................................................... 38
3.3.2 Between-line differences (HCR vs. LCR) ........................................ 40
3.3.3 Exercise effects (Exhaustion vs. Rest) ............................................ 41
3.3.4 Aging effects (Old vs. Young) ......................................................... 42
3.3.5 Pathway analyses of the three factors ............................................ 43
3.3.6 Interaction effects ............................................................................ 44
3.4 Discussion .................................................................................................... 45
3.5 Figures.......................................................................................................... 48
3.6 Tables ........................................................................................................... 53
CHAPTER 4: High Density SNP Array and Genome Sequencing Reveal Signatures
of Selection in a Divergent Selection Rat Model for Aerobic Running Capacity ... 56
4.1 Introduction ................................................................................................... 56
4.2 Materials and Methods ................................................................................. 57
4.2.1 Study overview ............................................................................... 57
4.2.2 Direction of comparisons ................................................................ 57
4.2.3 Genotyping data collection and quality control ................................ 58
4.2.4 Pooled whole-genome sequencing (WGS) and quality control ....... 59
4.2.5 Identification of long runs of homozygosity (ROH) .......................... 59
4.2.6 Fixation index (F ) .......................................................................... 61
st
4.2.7 Aberrant allele frequency spectrum (AFS) ...................................... 61
4.2.8 Composite score ............................................................................. 62
4.2.9 Pathway analysis ............................................................................ 64
4.2.10 Visualization of enriched pathways ............................................... 65
v
4.3 Results.......................................................................................................... 66
4.3.1 Runs of homozygosity (ROH) ......................................................... 66
4.3.2 Fixation index (F ) .......................................................................... 67
st
4.3.3 Aberrant allele frequency spectrum (AFS) ...................................... 66
4.3.4 Correlations among the three scan statistics .................................. 67
4.3.5 Overlap of top ranked genes among the three statistics ................. 68
4.3.6 Composite selection signature ........................................................ 69
4.4 Discussion .................................................................................................... 73
4.5 Figures.......................................................................................................... 76
4.6 Tables ........................................................................................................... 91
CHAPTER 5: F2-Based QTL Mapping ........................................................................ 96
5.1 Introduction ................................................................................................... 96
5.2 Materials and Methods ................................................................................. 97
5.2.1 “F2” intercross and phenotyping ..................................................... 97
5.2.2 SNP content design for the Affymetrix Axiom genotyping array ...... 98
5.2.3 Sample selection for genotyping ................................................... 100
5.2.4 Genotype data QC ........................................................................ 101
5.2.5 QTL mapping ............................................................................... 102
5.3 Results........................................................................................................ 102
5.3.1 "F2" Intercross of HCR-LCR ......................................................... 102
5.3.2 Genotyping data collection and QC .............................................. 104
5.3.3 QTL Mapping by association analysis ........................................... 104
5.4 Discussion .................................................................................................. 107
5.5 Figures........................................................................................................ 109
CHAPTER 6: Conclusions and Future Directions .................................................. 124
6.1 Genetic analysis of the pedigrees ............................................................... 124
6.2 Selection-, age-, and exercise-dependence of skeletal muscle gene
expression patterns .......................................................................................... 125
6.3 High-density SNP array and genome sequencing reveal signatures of
selection ........................................................................................................... 126
6.4 F2-based QTL mapping .............................................................................. 127
6.5 Next steps for causal gene identification .................................................... 128
REFERENCES ............................................................................................................ 129

vi
LIST OF TABLES

Table 2.1: Number of phenotyped animals (N) by line and generation, and the number
of animals chosen as effective breeders (N ), separately shown for male (N ) and
b m
female (N) ..................................................................................................................... 30
f
Table 2.2: Summary of cohort size, running distance, and body weight by gender and by
generation for HCRs ...................................................................................................... 31
Table 2.3: Summary of cohort size, running distance, and body weight by gender and by
generation for LCRs ...................................................................................................... 32
Table 3.1: -log(p-values) for the major pathway groups in the overall main effects of the
three factors .................................................................................................................. 53
Table 3.2: -log(p-values) for the major pathway groups in each main effect analysis. .. 54
Table 3.3: Directions of the major pathway groups ....................................................... 55
Table 4.1: Significant (p<0.0001) LRpath pathway results for composite HCR G26-G5
analysis ......................................................................................................................... 91
Table 4.2: Significant (p<0.0001) LRpath pathway results for composite LCR G26-G5
analysis ......................................................................................................................... 92
Table 4.3: 12 overlapping genes between the top 100 genes from the G5 and G26
HCR-LCR composite analyses ...................................................................................... 93
Table 4.4: Significant (p<0.0001) LRpath pathway results for composite G5 HCR-LCR
analysis ......................................................................................................................... 94
Table 4.5: Significant (p<0.0001) LRpath pathway results for composite G26 HCR-LCR
analysis ......................................................................................................................... 95

vii
LIST OF FIGURES

Figure 2.1: Distribution of predicted inbreeding coefficients (F) for G0 to G28 .............. 24
Figure 2.2: Distribution of maximal running distance for generations 0 to 28 ................ 25
Figure 2.3: Distribution of body weight for generations 0 to 28 for HCRs
(A) and LCRs (B) ........................................................................................................... 26
Figure 2.4: Progressive genetic differentiation revealed by 10K SNP genotyping
data ............................................................................................................................... 27
Figure 2.5: Decrease of average heterozygosity over time in both lines ....................... 28
Figure 2.6: Linkage disequilibrium (LD) decay over distance in HCR (A) and LCR (B) for
chromosome 1 .............................................................................................................. 29
Figure 3.1: Principal component analysis (PCA) plot (PC1 vs PC2) for 48 rats across the
expression of ~20K transcripts ...................................................................................... 48
Figure 3.2: Cube depiction of the PCA plot in Figure 3.1 .............................................. 49
Figure 3.3: Euclidean distances between each sample group ...................................... 50
Figure 3.4: Heatmap of significantly (p<0.001) differentially expressed genes for the
interaction effects represented by the left and right faces of the cube from Figure 3.2 . 52
Figure 4.1: Example tracks of the three statistics across Chromosome 1 for HCR G5
and G26 ........................................................................................................................ 75
Figure 4.2: Raw and transformed density plots of the three statistics ........................... 76
Figure 4.3: Distribution of ROH lengths across (a) HCR and (b) LCR groups ............... 77
Figure 4.4: Distribution of number of SNPs per ROH for (a) HCR and (b) LCR
groups ........................................................................................................................... 78
Figure 4.5: Distribution of gap length between ROHs for (a) HCR and (b) LCR
groups ........................................................................................................................... 79
Figure 4.6: Distribution of F values for (a) temporal and (b) between-line
st
comparisons .................................................................................................................. 80
Figure 4.7: Distribution of log(CLR) values per 1 Mb window for (a) HCR and (b) LCR
groups ........................................................................................................................... 81
Figure 4.8 Scatterplot between test statistic scores among HCR G26-G5 comparisons
for overlapping genes .................................................................................................... 82
Figure 4.9 Scatterplot between test statistic scores among LCR G26-G5 comparisons
for overlapping genes .................................................................................................... 83
Figure 4.10 Scatterplot between test statistic scores among G5 HCR-LCR comparisons
for overlapping genes .................................................................................................... 84
Figure 4.11 Scatterplot between test statistic scores among G26 HCR-LCR
comparisons for overlapping genes .............................................................................. 85
Figure 4.12: Manhattan plot of Composite Scores of Every Gene for HCR G26-G5 (a),
LCR G26-G5 (b), G5 HCR-LCR (c), and G26 HCR-LCR (d) ......................................... 86
viii
Figure 4.13: EnrichmentMap output for HCR G26-G5 LRpath results from the composite
analysis ......................................................................................................................... 87
Figure 4.14: EnrichmentMap output for LCR G26-G5 LRpath results from the composite
analysis ......................................................................................................................... 88
Figure 4.15: EnrichmentMap output for G5 HCR-LCR LRpath results from the
composite analysis ........................................................................................................ 89
Figure 4.16: EnrichmentMap output for G26 HCR-LCR LRpath results from the
composite analysis ........................................................................................................ 90
Figure 5.1: Distribution of running performance for animals of the F2 intercross
experiment .................................................................................................................. 109
Figure 5.2: Coverage of the ~625K SNPs ................................................................... 110
Figure 5.3: Intermarker distance (in Mb, y-axis) for each chromosome (x-axis) for the
~625K SNPs ................................................................................................................ 111
Figure 5.4: Z0-Z1 relatedness plot for all pairwise comparisons among (a) batch 1 and
(b) batch 2 samples ..................................................................................................... 112
Figure 5.5: GWAS results for linear model (a), linear model with family ID as covariate
(b), and EMMAX (b) across 381K SNPs in 616 F2 samples for the maximal running
distance trait ................................................................................................................ 114
Figure 5.6: Scatterplot of GWAS results for linear model (x-axis) without covariates (a)
and with family ID as covariate (b) versus EMMAX (y-axis) across 381K SNPs in 616 F2
samples for the maximal running distance trait ........................................................... 115
Figure 5.7: QQ-plot of GWAS p-values for linear model (green), linear model with family
ID as covariate (blue), and EMMAX (black) across 381K SNPs in 616 F2 samples for
the maximal running distance trait ............................................................................... 116
Figure 5.8: Manhattan zoom plot of 5 Mb up- and down-stream from the most significant
SNP (Chr6: 94770157) ................................................................................................ 118
Figure 5.9: Manhattan zoom plot of 5 Mb up- and down-stream from the most significant
SNP (Chr18: 12529014) with genes within the region below ...................................... 119
Figure 5.10: (a) Conditional GWAS results and (b) QQ-plot for EMMAX across 381K
SNPs in 616 F2 samples for the maximal running distance trait with the top SNP
(Chr18: 12529014) added as covariate ....................................................................... 120
Figure 5.11: Pooled WGS allele counts for the four groups (n=10 each) from Chapter 4
across the Dsc3 gene .................................................................................................. 121
Figure 5.12: Pooled WGS allele counts for the four groups (n=10 each) from Chapter 4
across the Dsc2 gene ................................................................................................. 122
Figure 5.13: Pooled WGS allele counts for the four groups (n=10 each) from Chapter 4
across the Dsg4 gene ................................................................................................ 123

ix