Antoine, Fort Publication

182 Pages·2017·5.43 MB·English

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Genome dosage and parent of origin effects in a ploidy series
Title
of Arabidopsis thaliana
Author(s) Antoine, Fort
Publication
2013-11-11
Date
Item record http://hdl.handle.net/10379/4432
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Genome Dosage and
Parent-of-Origin Effects
In a Ploidy Series of
Arabidopsis thaliana
Volume 1 of 1

Antoine Fort, BSc., MSc.

A thesis submitted to the National University of Ireland, Galway
For the degree of Doctor of Philosophy
College of Science, School of Natural Sciences
Discipline of Botany and Plant Science
Under the supervision of Prof. Charles Spillane
Head of School of Natural Sciences – Prof. Vincent O’Flaherty
December 2013
Table of Contents

Table of Contents
Table of Contents.............................................................................. 1

DECLARATION .................................................................................. 6

ACKNOWLEDGEMENTS ...................................................................... 7

LIST OF PUBLICATIONS AND CONFERENCES .................................... 8
PUBLICATIONS: ...................................................................................... 8
ORAL PRESENTATIONS: ........................................................................... 9

SUMMARY OF THE CONTENTS ......................................................... 10

INTRODUCTION .............................................................................. 11
Polyploidy in plants and animals, an overview .................................... 11
Evolutionary implications of polyploidy in plants ................................. 14
Formation of new species through polyploidy ............................................. 14
Genome evolution following polyploidy ...................................................... 16
Gene evolution following polyploidy .......................................................... 18
Is more the better? Applied potential of polyploid plants ...................... 20
Polyploidy in the “omics” era ............................................................ 22

CHAPTER 1 – ................................................................................... 24
PHENOTYPIC AND TRANSCRIPTOMIC DIFFERENCES IN ISOGENIC
POLYPLOIDS OF ARABIDOPSIS THALIANA ..................................... 24
INTRODUCTION .............................................................................. 25
RESULTS ....................................................................................... 27
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Table of Contents

Seed size differences in Arabidopsis C24 ploidy series. ................................ 27
Paternal excess F1 triploids displays increased biomass ............................... 28
Circadian clock-associated genes show differential amplitude between
reciprocal triploids. ................................................................................. 30
Differential tolerance to abiotic stress in isogenic F1 triploids ....................... 34
Parent of origin effects on stress response persist in mature Arabidopsis
reciprocal F1 triploids .............................................................................. 36
Alteration of autopolyploid gene expression in response to abiotic stress ....... 39
The transcriptional response to triploidy is strongly affected by cross direction
(i.e. a parental effect). ............................................................................ 41
Genes dysregulated by ploidy and parent-of-origin effects in F1 triploids are
enriched for stress-response genes ........................................................... 42
DISCUSSION .................................................................................. 44
Triploids display heterotic traits without genetic variation ............................ 44
Significant parent-of-origin effects on gene expression between reciprocal F1
triploids that are isogenic. ....................................................................... 45
MATERIALS AND METHODS .............................................................. 48
Plant growth and culture ......................................................................... 48
Abiotic stresses ...................................................................................... 48
Microarray analysis ................................................................................. 49
qRT-PCR expression analysis .................................................................... 50

CHAPTER 2 – ................................................................................... 51
TRANSCRIPTOME AND METHYLOME PROFILING OF ISOGENIC
POLYPLOID LINES OF Col-0 ARABIDOPSIS THALIANA ................... 51
Abstract......................................................................................... 52
1. Introduction ............................................................................... 52
2. Materials and Methods ................................................................. 54
2.1 Plant materials .............................................................................. 54
2.2 Sample preparation and microarray hybridization .............................. 54
2.3 Tiling array and small RNA data analysis .......................................... 55
S2.1 Microarray data analysis .............................................................. 55
S2.2 Gene expression......................................................................... 56
S2.3 DNA methylation ........................................................................ 56
S2.4 Identification of indels ................................................................. 57
S2.5 Correlation between DNA methylation and gene expression ............. 57
S2.6 Gene expression quantification ..................................................... 57
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S2.7 Small RNA mapping .................................................................... 58
3.Results and discussion .................................................................. 58
3.1 Generation of isogenic F1 triploid plants ........................................... 58
3.2 High-density tiling array analysis confirms that isogenic reciprocal F1
triploids generated from inter-ploidy crosses in Arabidopsis thaliana are
genetically identical ................................................................................ 59
3.3 Isogenic reciprocal F1 triploid plants display epigenetic parent-of-origin
specific genome dosage effects on transcript levels ..................................... 60
3.4 The 602 genes subject to parent-of-origin specific genome dosage
effects are enriched for stress response genes ........................................... 62
3.5 Paternal excess isogenic F1 triploids display elevated mCG methylation at
CCGG sites ............................................................................................ 63
3.6 Parental genome dosage effect polymorphic CmCGG methylation is
uniformly distributed across chromosomal regions in reciprocal F1 triploids.... 64
3.7 Polymorphic CmCGG methylation is not associated with any particular
genomic feature in the reciprocal F1 triploids. ............................................ 67
3.8 Constitutive (but not polymorphic) CmCGG gene body methylation in F1
triploids displays similar patterns to diploids .............................................. 68
3.9 Gene expression differences between reciprocal F1 isogenic triploids is not
correlated with differential CmCGG methylation ........................................... 71
3.10 24nt small RNAs differentially accumulate in up and downstream regions72
3.11 Conclusions .................................................................................... 78

CHAPTER 3 – ................................................................................... 79
ALLELE-SPECIFIC GENE EXPRESSION IN TRIPLOID F1 HYBRIDS OF
ARABIDOPSIS THALIANA ............................................................... 79
INTRODUCTION .............................................................................. 80
RESULTS ....................................................................................... 82
Gene dysregulation in isogenic reciprocal F1 triploids persists in triploid F1
hybrid context ........................................................................................ 82
Identification of parental contributions to the overall gene expression in
reciprocal triploid F1 hybrids by Sanger sequencing. ................................... 82
Validation of the maternal allele expression bias by QUASEP ........................ 87
Whole genome investigation of gene expression and parental contributions of
the transcriptome of reciprocal F1 hybrid triploid embryos. .......................... 89
780 genes are dysregulated between maternal and paternal excess F1 triploid
embryos. ............................................................................................... 90
Parental allele contributions to the entire transcriptome of reciprocal triploid Fq
hybrid embryos. ..................................................................................... 92
DISCUSSION ................................................................................ 100
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Paternal excess F1 triploid seedlings display increased expression of the haploid
maternal allele ..................................................................................... 100
Gene expression dysregulation between reciprocal F1 hybrid triploids. ........ 100
The unbalanced parental genome contribution in F1 triploid tissues is
responsible for triggering extensive allele-specific genomic imprinting ......... 102
Increased expression of the haploid maternally inherited allele expression in
paternal excess triploids ........................................................................ 103
Detection of 49 genes displaying gene-specific imprinting effects in reciprocal
F1 triploid embryos ............................................................................... 103
CONCLUSION ............................................................................... 105
MATERIALS AND METHODS ............................................................ 106
Plant material and growth conditions....................................................... 106
RNA extraction and qRT-PCR analysis ...................................................... 106
QUASEP analysis .................................................................................. 106
RNA-sequencing analysis ....................................................................... 107

CHAPTER 4 – ................................................................................. 109
PLOIDY EFFECTS ON BIOTIC STRESS TOLERANCE IN ARABIDOPSIS
THALIANA ..................................................................................... 109
INTRODUCTION ............................................................................ 110
RESULTS ..................................................................................... 111
Differential response to Botrytis cinerea infection in Arabidopsis autopolyploids111
Gene expression differences following Botrytis infection in C24 diploid and
tetraploid leaves. .................................................................................. 114
DISCUSSION ................................................................................ 117
CONCLUSION ............................................................................... 118
MATERIALS AND METHODS ............................................................ 119
Biotic stress analysis ............................................................................. 119
qRT-PCR analysis .................................................................................. 119

CHAPTER 5 – ................................................................................. 120
INCOMPATIBLE INTERPLOIDY CROSSES IN ARABIDOPSIS
THALIANA REQUIRE DNA DEMETHYLASES .................................... 120
INTRODUCTION ............................................................................ 121
RESULTS ..................................................................................... 123
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Table of Contents

The triploid block in Arabidopsis thaliana is accession specific..................... 123
Epigenetic modifiers are dysregulated in Paternal Excess triploid seedlings. . 124
Genotyping of ros1-3;dml2-1;dml3-1 (rdd) triple mutant. ......................... 126
The loss of function rdd triple mutant overcomes the triploid block in inter-
ploidy crosses. ..................................................................................... 127
Seed size is not altered in rdd paternal excess triploids. ............................ 130
rdd mutation suppresses the low germination phenotype of paternal excess F1
triploid seeds. ...................................................................................... 132
ROS1 is the most highly expressed DEMETER-LIKE gene in developing seeds.136
ROS1 is under maternal control. ............................................................. 137
Triploid block-associated dysregulation of the FIS complex is suppressed in rdd
paternal excess triploid F1 seeds. ........................................................... 139
Pathogen response genes are up-regulated in response to interploidy crosses.141
DISCUSSION ................................................................................ 144
Link between DNA methylation and interploidy crosses incompatibility. ....... 144
rdd suppresses the dysregulation of PRC2 genes and targets in incompatible
interploidy crosses ................................................................................ 144
rdd-dependent immune response activation during interploidy crosses ........ 145
MATERIALS AND METHODS ............................................................ 147
Plant materials and growth conditions. .................................................... 147
Analysis of seed phenotype .................................................................... 147
qRT-PCR analysis .................................................................................. 148

GENERAL CONCLUSION AND FUTURE DIRECTIONS....................... 150
Heterosis without hybridity (SNPs): Arabidopsis thaliana reciprocal F1
triploids as an emerging model for epigenetic heterosis ..................... 150
New insights into the molecular basis of incompatible interploidy crosses154

TABLE OF FIGURES ....................................................................... 156

REFERENCES ................................................................................. 165

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DECLARATION

DECLARATION
I certify that this thesis is my own work, and that I have not used this work in the
course of another degree, either at National University of Ireland Galway, or
elsewhere.

Signed: ____________________
Antoine Fort

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ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

To my friends and colleagues, from Ireland or back home, that made this Ph.D an
almost every day pleasure,
To Dr Peter, Dr Mark and Dr Duygu for their help and friendship,
To Michel, Martin, Udo and Guillaume for showing me the dark side(s) of Galway,
To Charlie for the continuous help,
To the Scholar’s Rest for providing the much needed beer at the end of the days,
To my parents, and to Niamh, obviously,
Thank you.

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LIST OF PUBLICATIONS AND CONFERENCES

LIST OF PUBLICATIONS AND CONFERENCES
PUBLICATIONS:

2013:
Donoghue MTA, Fort A, Clifton R, Xhang X, McKeown PC, Borevitz JO and Spillane
C (2013): CmCGG-methylation independent parent-of-origin effects on
genome-wide transcript levels in isogenic reciprocal F1 triploid plants. DNA
Research (in press)
McKeown PC, Fort A, Duszynska D, Sulpice R and Spillane C (2013) Emerging
molecular mechanisms for biotechnological harnessing of heterosis in
crops. Trends in Biotechnology 31:549-51
McKeown PC, Fort A and Spillane C (2013): Analysis of genomic imprinting by
quantitative allele-specific expression by pyrosequencing®. In: McKeown PC
and Spillane C (Eds) (2013) Treasuring Exceptions: Plant Epigenetics and
Epigenomics. Humana Press: New Jersey, USA. (in press)

2012:
Selcuklu S, Donoghue MTA, Rehmet K, de Souza Gomes M, Fort A, Kovvuru P,
Muniyappa M, Kerin M, Enright A, Spillane C. MicroRNA-9 inhibition of cell
proliferation and identification of novel miR-9 targets by transcriptome
profiling in breast cancer cells. Journal of Biological Chemistry 287:29516-28.
McKeown PC, Keshaviaiah C, Fort A, Tuteja R, Chatterjee M, Varshney RK and
Spillane C. Harnessing Genomics To Improve Nutritional Content And
Bioavailability Of Plant-Derived Foods, in the book P.S. Panesar and S.S.
Marwaha (2012). “Biotechnology in Agriculture and Food Processing: Opportunities
and Challenges"
McKeown P, Fort A and Spillane C (2012) Genomic imprinting: Parental control
of gene expression in higher plants. In: Birchler J and Chen ZJ (Eds.) Plant
Polyploidy and Hybrid Genomics, John Wiley & Sons Ltd (in press).
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