rDNA Copy Number Change and Sequence Variation in Mutation Accumulation Lines of Daphnia ...

217 Pages·2017·3.63 MB·English

by  Eleanor Harvey

rDNA Copy Number Change and Sequence Variation in Mutation
Accumulation Lines of Daphnia pulex

by

Eleanor Floy Harvey

A Thesis
Presented to
The University of Guelph

In partial fulfillment of requirements
for the degree of
Master of Science
in
Integrative Biology

Guelph, Ontario, Canada
© Eleanor Floy Harvey, January, 2018
ABSTRACT

rDNA Copy Number Change and Sequence Variation in Mutation Accumulation Lines of
Daphnia pulex

Eleanor Floy Harvey Advisor:
University of Guelph, 2018 Dr. Teresa J. Crease

Freshwater ecosystems are subject to a variety of stressors, including metal
contamination. Lakes and ponds in southern Ontario are especially contaminated with
metals, as mines in this region are among the top copper and nickel producers in the
world. These metals can mutate DNA, although it is generally unknown how the
freshwater microcrustacean Daphnia pulex is affected by chronic metal exposure. To
determine how metal exposure affects D. pulex ribosomal (r)DNA, a highly conserved
genetic compartment, I estimated rDNA copy number and sequence variation in mutation
accumulation lines of D. pulex. I found that chronic metal exposure results in large
fluctuations in rDNA copy number in D. pulex, including significant losses of rDNA
copies. However, chronic metal stress does not influence the rate of new point mutations
in D. pulex rDNA. These findings contribute to our understanding of how D. pulex rDNA
evolves with chronic, sub-lethal exposure to metal.
ACKNOWLDEGEMENTS

I would like to extend my sincerest gratitude to Dr. Teresa Crease for her constant
and unwavering assistance and patience, and for indulging me with her encyclopedic
knowledge of virtually everything. I am beyond grateful for the opportunity to learn from
her and work in her lab, and am much wiser and more fulfilled now than when I arrived
in Guelph, thanks to her mentorship.
I would also like to thank the members of my Advisory Committee, Dr.
Melania Cristescu, Dr. Elizabeth Boulding, and Dr. Lewis Lukens, for their time, advice,
and invaluable feedback about my research. I owe Dr. Melania Cristescu particular
gratitude for acting as my second de facto supervisor. Not only did her lab generously
host me while I collected my Daphnia samples, she also asked me thought-provoking
questions that helped to guide the direction of my research objectives. I am also therefore
indebted to many current and previous members of the Cristescu lab at McGill
University, including Dr. Frédéric Chain, Tiffany Chin, James Bull, Sarah Finlayson,
and Jullien Flynn, who were always patient with me, and who provided friendship and
entertainment during the many conferences and workshops that we attended together. In
particular, I would like to thank Dr. Frédéric Chain who was the Postdoctoral student in
the Cristescu lab for most for my degree, for all of his valuable bioinformatics guidance,
understanding, and alacrity.
I am endlessly thankful to Dr. Tyler Elliot for his uncompromised patience,
generosity of time, mentorship, and willingness to help in and out of the lab. He truly
enriched my time at the University of Guelph with his wisdom and humor. I also owe the
University of Guelph Genomics Facility, especially Jing Zhang and Angela Holliss, my
thanks for their help, advice, and expeditious turnaround time of my data. Furthermore, I
could not have completed this project without the data in this thesis and in related papers
that was painstakingly collected over many long hours by Michelle Hotchkiss, Jenna
Dale, Hailey Hunter, and Connor Randall. I am grateful to numerous other graduate
students in the Department of Integrative Biology for their stimulating conversation,
perspectives, support, and friendship, including Darren Kelly, Mason Stothart, Carolyn
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Trombley, Caleb Axelrod, Katherine Drotos, Haley Yorke, Tony Kess, Brent Saylor, and
many others.
Finally, I could not have completed this thesis without the help of my family and
friends, most of whom hail from the United States. Thank you to Dorothy Bakker for
encouraging my personal growth outside of my schoolwork. Granny, thank you for
insight into and interest in my work. Thank you to my oldest friends Maria Cuneo and
Roger LeBlanc for your love, your caring, and for your reminders to visit more often.
Finally, thank you Mom and Dad for your unending support in my crazy ideas, and for
your unwavering trust in me that I’ll get the job done. I dedicate this thesis to you all.

This project was supported in part by an NSERC CREATE grant for Aquatic
Ecosystem Health created by Dr. Melania Cristescu.

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STATEMENT OF AUTHORSHIP and CONTRIBUTIONS

I am the main author of this thesis and of the manuscripts contained within it. I
conducted the end-point PCR tests with the help of Michelle Hotchkiss and I conducted
the quantitative PCR tests with the help of Hailey Hunter, Jenna Dale, and Connor
Randall. I analyzed the whole genome sequencing data provided by the Cristescu lab with
the help of Dr. Frédéric Chain. My advisors, Dr. Teresa J. Crease and Dr. Melania
Cristescu are primarily responsible for the development of this project and for its
experimental design.

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TABLE of CONTENTS
ABSTRACT ........................................................................................................................ ii
STATEMENT OF AUTHORSHIP and CONTRIBUTIONS ............................................ v
LIST of TABLES .............................................................................................................. vii
LIST of FIGURES ............................................................................................................. ix
LIST of APPENDICES...................................................................................................... xi
CHAPTER 1 ....................................................................................................................... 1
Metal pollution in North American freshwater ecosystems ........................................... 2
Study Species: Daphnia pulex ........................................................................................ 4
The effects of copper and nickel on the genome ............................................................ 8
Daphnia pulex and mutation accumulation experiments .............................................. 10
Ribosomal DNA............................................................................................................ 12
Objectives of this study ................................................................................................. 15
CHAPTER 2 ..................................................................................................................... 17
Abstract ......................................................................................................................... 17
Introduction ................................................................................................................... 18
Methods......................................................................................................................... 21
Results ........................................................................................................................... 28
Discussion ..................................................................................................................... 32
Conclusions and Future Work ...................................................................................... 41
CHAPTER 3 ..................................................................................................................... 42
Abstract ......................................................................................................................... 42
Introduction ................................................................................................................... 43
Methods......................................................................................................................... 46
Results ........................................................................................................................... 50
Discussion ..................................................................................................................... 56
Conclusions and Future Work ...................................................................................... 68
CHAPTER 4 ..................................................................................................................... 70
REFERENCES ................................................................................................................. 74
TABLES ........................................................................................................................... 86
FIGURES ........................................................................................................................ 123
APPENDICES ................................................................................................................ 147

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LIST of TABLES

Table 1. Primers used in PCR and qPCR experiments ..................................................... 86

Table 2. Mean haploid 18S gene copy number and rate of copy number change (Δ Copy
number) in the obligately parthenogenetic, clonal hybrid (CH) lineage .......................... 87

Table 3. Mean haploid 18S gene copy number and rate of copy number change (Δ Copy
number) in the cyclically parthenogenetic, sexual (SX) lineage ...................................... 89

Table 4. P-values from Kruskall-Wallis tests for differences in mean haploid 18S gene
copy number among treatments for both the CH and SX lineages ................................... 91

Table 5. P-values from Mann-Whitney Tests for differences in mean haploid 18S gene
copy number and copy number rate of change (Δ Copy number) between the CH and SX
lineages ............................................................................................................................. 92

Table 6. P-values from Mann-Whitney Tests for differences in mean haploid 18S gene
copy number and copy number rate of change (Δ Copy number) between treatments for
both the CH and SX lineages ............................................................................................ 94

Table 7. P-values from Kruskall-Wallis tests of differences in mean haploid 18S gene
copy number between generations, within treatments, for both the CH and SX lineages 97

Table 8. P-values from Mann-Whitney Tests for differences in mean haploid 18S gene
copy number between generations, within treatments, for both the CH and SX lineages 98

Table 9. P-values from Levene's Test for tests of differences in haploid 18S gene copy
number variance among treatments, for both the CH and SX lineages ............................ 99

Table 10. All point mutations (PMs), corresponding mutation rates, and standard error
(SE) of mutation rates for all treatments and rDNA regions of the CH lineage ............. 100

Table 11. All point mutations (PMs), corresponding mutation rates, and standard error
(SE) of mutation rates for all treatments and rDNA regions of the SX lineage ............. 101

Table 12. P-values from Mann-Whitney tests for differences in average rDNA point
mutation rate between the CH and SX lineages.............................................................. 102

Table 13. P-values from Kruskall-Wallis (KW) tests for differences in mean point
mutation rate between regions of rDNA and treatments, for both the CH and SX lineages
......................................................................................................................................... 103

Table 14. R2 values, correlation coefficients from Pearson’s product-moment correlation
and corresponding p-values between the mean 18S gene copy number and the mean point
mutation rate across regions of the rDNA repeat for both the CH and SX lineages ...... 105
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Table 15. Spectrum of point mutations (PMs) sustained in Daphnia mutation
accumulation lines .......................................................................................................... 106

Table 16. rDNA mutation rates for each category of nucleotide transition or transversion
event, and corresponding standard errors (SE) of mutation rates ................................... 108

Table 17. All indel mutations (IMs) that occurred in Daphnia mutation accumulation
lines ................................................................................................................................. 109

Table 18. Variants in rDNA of the CH and SX lineages and the pCH individuals ........ 110

Table 19. Mean variant per kB of rDNA and frequency of variants for all rDNA regions
and treatments, for both the CH and SX lineages ........................................................... 111

Table 20. P-values from Kruskall-Wallis tests for differences in mean number of variants
per kB of rDNA and mean frequency of variants among rDNA regions and treatments,
for both the CH and SX lineages .................................................................................... 113

Table 21. P-values from Mann-Whitney tests for differences in mean variant per kB of
rDNA and mean frequency of variants for the CH lineage ............................................ 114

Table 22. P-values from Mann-Whitney tests for differences in mean variant per kB of
rDNA and mean frequency of variants in the SX lineage .............................................. 117

Table 23. P-values from Mann-Whitney tests for differences in mean variant per kB of
rDNA and mean frequency of variants between the CH and SX lineages ..................... 119

Table 24. Variance of variant frequency across samples ................................................ 120

Table 25. P-values between variant frequency and variance for high and low frequency
variants in the CH and SX lineages from Wilcox rank sum tests with continuity
correction ........................................................................................................................ 121

Table 26. Correlation coefficients from Pearson’s product-moment correlation and
corresponding p-values between rDNA copy number estimates from qPCR and estimates
based on sequence read depth ......................................................................................... 122

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LIST of FIGURES

Figure 1. Diagram of a eukaryotic ribosomal DNA (rDNA) repeat ............................... 123

Figure 2. Distribution of MA samples used in all qPCR experiments among treatments
and generations ............................................................................................................... 124

Figure 3. Mean haploid 18S gene copy number in the control samples and pCH
(population of CH) individuals for all generations of the CH (clonal hybrid) and SX
(sexual) lineages.............................................................................................................. 125

Figure 4. Mean haploid 18S gene copy number and rate of copy number change for all
generations in the CH lineage ......................................................................................... 126

Figure 5. Mean haploid 18S gene copy number and rate of copy number change for all
generations in the SX lineage ......................................................................................... 127

Figure 6. Mean haploid 18S gene copy number for generations 45, 70, and 100 in the CH
lineage ............................................................................................................................. 128

Figure 7. Mean haploid 18S gene copy number for generations 45, 70, and 100 in the SX
lineage ............................................................................................................................. 129

Figure 8. Distribution of MA samples used in all sequence variation analyses among
treatments and generations .............................................................................................. 130

Figure 9. Distribution of the 56 point mutations (PMs) in the rDNA of the 55 sequenced
samples of the CH lineage .............................................................................................. 131

Figure 10. Location in the rDNA and frequency of the 56 point mutations (PMs) in the 55
sequenced samples of the CH lineage ............................................................................. 132

Figure 11. Distribution of the 41 point mutations (PMs) in the rDNA of the 36 sequenced
samples of the SX lineage ............................................................................................... 133

Figure 12. Location in the rDNA and frequency of the 41 point mutations (PMs) in the 36
sequenced samples of the SX lineage ............................................................................. 134

Figure 13. Point mutation rates in the treatments of the CH and SX lineages ............... 135

Figure 14. Point mutation rates in the rDNA regions of the CH and SX lineages ......... 136

Figure 15. Relationship between rDNA point mutations and mutation rate and 18S rRNA
gene copy number ........................................................................................................... 137

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Figure 16. Point mutation spectrum in the rDNA of mutation accumulation (MA) lines of
Daphnia........................................................................................................................... 138

Figure 17. Location in the rDNA and frequency of variant sites across all samples in the
CH and SX lineages and in the pCH (population of CH) individuals ............................ 139

Figure 18. Distribution of variants among samples in the CH and SX lineages ............ 140

Figure 19. Number of variants per kB of rDNA among rDNA regions and within
treatments in the CH and SX lineages and in the pCH (population of CH) individuals. 141

Figure 20. Number of variants per kB of rDNA among treatments and across all rDNA
regions in the CH and SX lineages ................................................................................. 142

Figure 21. Mean frequency of variants in each treatment, within rDNA regions in the CH
and SX lineages............................................................................................................... 143

Figure 22. Mean frequency of variants among rDNA regions and within treatments in the
CH and SX lineages and in the pCH individuals ............................................................ 144

Figure 23. Mean frequency of indel variants (IVs) and point variants (PVs) across all
treatments and rDNA regions in the CH and SX lineages and in the pCH individuals .. 145

Figure 24. Mean frequency of indel variants (IVs) and point variants (PVs), within rDNA
regions in the CH and SX lineages ................................................................................. 146

x