Table Of ContentGenomes in the evolution of polyploid crop species and hybrids
Thesis submitted for the degree of
Doctor of Philosophy
at the University of Leicester
by
Farah Badakshi
Department of Biology
University of Leicester
December 2013
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Abstract
Genomes in the evolution of polyploid crop species and hybrids
Farah Badakshi
Many of the world’s crop species are recent polyploids. The various genomes
from the diploid ancestors (known or, often, unknown) interact, with variable
effects on genome packaging and nuclear organization (together the nuclear
architecture), chromosome stability and gene expression. This project used a
comparative approach to understand the genome composition in polyploids,
focusing on millets in the Panicum group, saffron Crocus, Brassica and
Nicotiana. In situ hybridization using DNA probes was used to identify the
chromosomes and antibodies to synaptonemal complex, DNA repair and
chromatin structure proteins including histones, which allow the understanding
of the modulation of chromosome behaviour depending on the ancestral origin
of the chromosomes, were used.
The ancestors of proso millet, P. miliaceum (2n=4x=36), were identified as P.
capillare and being the same as one genome in the 4x P. repens by in situ
hybridization and ITS sequencing. A cell fusion hybrid of Nicotiana x sanderae +
N. debneyi was confirmed, with demonstration of chromosome loss, by IRAP
markers and in situ hybridization. Saffron Crocus, Crocus sativus 2n=3x=24,
was shown to not be an autopolyploid, but to include three genomes with
somewhat different chromosomal and sequence characteristics. The alien lines
of Brassica and Raphanus with the fertility restorer genes were identified with B.
rapa carrying the two chromosomes of Raphanus carrying the fertility restorer
genes. Furthermore, the meiotic pairing basis of the alien lines of Brassica and
Orychophragmus was also observed which gives an insight into the meiotic
pairing between two different species. The water stress resistant genes could
be identified from Panicum and thus be utilized in better water usage of plants.
It would be possible in future to develop a synthetic C. sativus and thus rescue
its declining production around the world, thus improving its economic potential.
The fertility restorer gene can now be introduced into the B. rapa species using
various mutagens.
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Acknowledgements
I would never have been able to finish my dissertation without the guidance of
my committee members, help from friends, and support from my family.
I would like to express my deepest gratitude to my supervisor Prof JS Heslop
Harrison, for giving me an opportunity to do my PhD under his excellent
guidance, providing me with an excellent atmosphere for doing research and
being my mentor. I would also like to thank Dr Trude Schwarzacher, Dr John
Bailey for being a constant support in the lab, and Dr Sinead Drea for being my
internal accessor and, giving her useful advice. I would also like to thank all the
colleagues I worked with for their help and guidance. I take this opportunity to
thank all the collaborators I worked with for accepting me as a part of their
project.
Nothing would have been possible without the support of my family. I would like
to thank my mother for her constant prayers, for being there all my life as a
constant support especially during my thesis writing period. To my dearest
father whose presence I still deeply miss, for being the motivation to do this
work and make his soul happy. Nothing would have been possible without the
constant moral support from my husband, being always there for me at difficult
times. Last but not the least I am thankful to my adorable little daughter for
being patient enough for letting me finish the thesis during the time that
belonged to her.
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Table of Contents
Abstract 2
Acknowledgements 3
Abbreviations 9
Chapter 1: Introduction to genomes in the evolution of polyploids 11
1.1 Speciation and polyploidy 11
1.1.1 Cytogenetics and karyotype evolution 13
1.1.2 Palaeopolyploids and neopolyploids 17
1.1.3 Chromosome doubling and genome size evolution 19
1.2 DNA sequences in the genome 22
1.2.1 Transposons and repeat sequences and their impact in polyploids 23
1.2.2 Chloroplast and nuclear genome 25
1.2.3 Molecular markers and evolution 27
1.3 Fluorescent in situ hybridization (FISH) 28
1.4 Evolution and phylogeny of angiosperms 30
1.5 Epigenetic makeover of polyploids 31
1.5.1 Nucleolar dominance 32
1.5.3 Meiotic behaviour in polyploids 34
1.6 Advantages and disadvantages of polyploidy 36
1.7 Polyploidy, domestication and crop evolution 39
1.8 Applications of polyploidy 43
1.9 Summary 44
1.10 Aims and objectives 46
Chapter 2: Materials and methods 48
2.1 Materials 48
2.2 Methods 48
2.2.1 Isolation of genomic DNA 48
2.2.2 DNA Quantification 49
2.2.3 Agarose gel electrophoresis 50
2.3 PCR 50
2.3.1 PCR markers 51
2.3.2 Purification of PCR products 51
2.3.3 Sequencing of PCR amplicons and sequence analysis 53
2.4 Probes used 53
2.4.1 Probes Labelling 54
2.4.1.1 PCR Labelling 54
2.4.1.2 Random primers labelling 54
2.4.2 Testing of labelled probes 55
2.5 Collection and fixation of root tips 57
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2.6 Chromosome preparation 58
2.6.1 Mitotic spreads 58
2.6.2 Preparation of meiotic spreads and sections 59
2.6.3 Staining chromosomes 59
2.6.3.1 Silver staining 60
2.7 Immunostaining 61
2.8 Fluorescent in situ hybridization 62
2.8.1 Pre-hybridization 62
2.8.2 Hybridization 63
2.8.3 Post hybridization 64
2.8.3 Slide detection 64
2.8.4 Slide mounting 65
2.8.5 Photography 65
Chapter 3: Origin and evolution of Panicum miliaceum (Proso millet) 67
3.1 Introduction 67
3.1.1 Origin and evolution 69
3.1.2 Phylogeny 69
3.1.2 Epigenetic modifications 71
3.1.3 Proso millet 71
3.1.3.1 Nutritional content and quality 73
3.1.4 Cytology and distribution 73
3.1.5 C4 Photosynthetic pathway 75
3.1.6 Nuclear and chloroplast genes 76
3.2 Materials 77
3.3 Methods 79
3.4 Results 79
3.4.1 In situ hybridization 79
3.4.2Immunostaining with anti-5-methylcytosine 85
3.4.3 Molecular marker analysis 87
3.4.3.1 Chloroplast and nuclear gene markers 87
3.4.3.2 Stress genes and NAD-ME malic acid 107
3.5 Discussion 109
3.5.1 Origin of genomes by genomic in situ hybridization 109
3.5.2 Nuclear genes in diploids and tetraploid Panicum 112
3.5.3 45S rDNA evolution in the tetraploid P. miliaceum 113
3.5.4 Meiosis in Panicum 115
3.5.5 DNA methylation 115
3.5.6 C4 Photosynthetic pathway 116
Chapter 4: Origin and evolution of the triploid Crocus sativus (saffron) 119
4.1 Introduction 119
4.1.1 Crocus sativus 119
4.1.2 Crocus evolution 120
4.1.3 Geographical distribution 123
4.1.4 Uses of saffron 124
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4.2 Cytology 124
4.2.1 C. sativus 124
4.2.2 Simple sequence repeats (SSRs) 125
4.3 Phylogeny 126
4.4 Materials 127
4.5 Methods 128
4.6 Results 128
4.6.1 Fluorescent in situ hybridization 128
4.6.1.1 C. cartwrightianus 134
4.6.1.2 C. thomasii 135
4.6.1.3 C. oreocreticus 137
4.6.1.4 C. hadriaticus 137
4.6.1.5 C. asumanae 137
4.6.1.6 C. pallasii 138
4.6.1.7 C. mathewii 138
4.6.2 Meiotic behaviour 140
4.6.3 Immunostaining 145
4.6.4 Crocus DNA markers 150
4.6.4.1 390F+1326R matK plastid gene sequence 150
4.6.4.2 XF +5R matK plastid gene sequence 151
4.6.4.3 1F+724R rbcl plastid gene sequence 151
4.6.4.4 TrnH plastid gene sequence 152
4.7 Discussion 158
4.7.1 In situ hybridization 158
4.7.2 Karyotype 159
4.7.3 Meiosis 160
4.7.4 Chloroplast sequences 163
Chapter 5: Identification of genomes in the tetraploid Nicotiana debneyi
and octaploid somatic hybrids with Nicotiana x sanderae 164
5.1 Introduction 164
5.1.1 rDNA Evolution 164
5.1.2 Alien introgression 165
5.1.2.1 Modes of Introgression 166
5.1.3 Parent plant significance and morphology 168
5.1.3.1 Nicotiana x sanderae 168
5.1.3.2 Nicotiana debneyi 168
5.1.4 Current Study 169
5.2 Materials 170
5.3 Methods 171
5.4 Results 171
5.4.1 Cytology and molecular analysis of regenerated putative somatic hybrid
plants. 171
5.4.2 Molecular analysis of DNA 175
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5.5 Discussion 176
Chapter 6: Alien chromosome introgression in Brassica species 181
6.1 Introduction 181
6.1.1 Brassica 181
6.1.2 Alien introgression 182
6.1.2.1 Identification of alien fragments 184
6.1.2.2 Brassica napus - Orychophragmus violaecus addition and
substitution lines 184
6.1.2.3 Brassica rapa – Raphanus sativus addition line 187
6.1.3 Meiotic analysis of chromosome pairing and Synaptonemal Complex
(SC) formation 189
6.1.3.1 SC proteins 191
6.1.4 Epigenetic modification 193
6.2 Section 1: Brassica rapa – Raphanus sativus addition line 194
6.2.1 Materials 194
6.2.1.1 Plant material 194
6.2.1.2 Probes used 195
6.2.2 Methods 195
6.2.3 Results 195
6.2.3 Discussion 199
6.3 Section 2: Brassica-Orycophragmus addition lines: 202
6.3.1 Materials 202
6.3.1.1 Plant materials 202
6.3.2 Method 202
6.3.3 Meiotic results 202
6.3.3.1 γH2AX collocalized with the Asy-1 preferentially on the unpaired
chromatin 208
6.3.4 DNA methylation 211
6.3.5 Discussion 213
6.3.5.1 DNA methylation 217
Chapter 7: Conclusions 219
7.1 Conclusion structure 219
7.2 Origin of polyploid species 219
7.3 Genome and chromosome transfer in hybrids 221
7.3.1 Somatic hybrids 221
7.3.2. Raphanus introgression into Brassica 221
7.4 Advantages of molecular cytogenetics investigations 222
7.5 Meiosis and recombination control 223
7.6 Future developments of better epigenetic control 225
7.7 Breeding using hybrids 226
7.8 Genome organization and evolution past and future 228
Appendix 2.1 229
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Appendix 3.1 231
Appendix 3.2 251
References 252
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Abbreviations
AFLP Amplified fragment length polymorphism
BAC Bacterial Artificial Chromosome
BCIP 5-bromo-4-chloro-3-indolyl-phosphate
bp Base pairs
BLAST Basic local alignment search tool
CTAB Cetyltrimethylammonium bromide
DAPI 4’,6-diamidino-2-phenylindole
dUTP Deoxyuridine triphosphate
DNA Deoxyribbonucleic acid
dNTP’s Deoxynucleotide triphosphate
EDTA Ethylenediammine tetra- acetic acid
FISH Fluoroscent in situ hybridization
GISH Genomic in situ hybridization
HCL Hydrochloric acid
Indels Insertions- deletions
INT 2- (4-iodophenyl)-5-(4-nitrophenyl)-3-phenyltetrazolium chloride
IRAP Inter-retroelement insertion polymorphism
ITS Internal transcribed spacers
LTRs Long terminal repeats
Mat k Maturase kinase
M Molar
Mg Miligram
ml Millilitre
9
mM Milimolar
µl Microlitre
µm Micromolar
MYA Million years ago
NOR Nuclear organizer region
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PHY C Phytochrome C
PVP Polyvinylpyrilidone
RAPD Random amplified polymorphic DNA
rbcl Ribulose-bisphosphate carboxylase gene
rDNA Ribosomal DNA
RFLP Restriction fragment length polymorphism
RNA Ribonucleic acid
RNAse Ribonuclease
rpm Rotations per minute
RT Room temperature
SC Synaptonemal Complex.
SDS Sodium dodecyl sulphate
Sec Seconds
SSC Saline sodium citrate
SSR’s Simple sequence repeats
v/v Volume added to volume
w/v Weight added to volume
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