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Novel genetic factors affecting bolting and floral transition control in Beta vulgaris [Elektronische Ressource] / vorgelegt von Salah Fatouh Abou-Elwafa

136 pages
Aus dem Institut für Pflanzenbau und Pflanzenzüchtung der Christian-Albrechts-Universität zu Kiel Novel Genetic Factors Affecting Bolting and Floral Transition Control in Beta vulgaris Dissertation zur Erlangung des Doktorgrades der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von M.Sc. Salah Fatouh Abou-Elwafa aus Sohag, Ägypten Kiel, 2010 Dekan: Professor Dr. Karin Schwarz 1. Berichterstatter: Professor Dr. Christian Jung 2. Berichterstatter: Professor Dr. Daguang Cai Tag der mündlichen Prüfung: 10.02.2010 Table of Contents I Tables of Contents Tables of Contents ………………………………………………………….……. I Abbreviations ………………………………………………………………… VList of Tables …………………………………………………………………. VIIList of Figures …………………………………………………………..……. VIII1. General introduction ………………………………………...…………... 11.1 History of sugar beet cultivation…………………………………….. 11.2 Biology, taxonomy and breeding of sugar beet ……………………… 11.3 Sugar beet cultivation in non-European countries …………………… 61.4 Genetic analysis of sugar beet ……………………………………….. 71.5 The impact of early bolting on sugar beet production ……………….. 81.6 Environmental and internal factors regulating bolting and flowering .. 91.7 Flowering time regulation in model species …………………………. 101.8 Flowering time genes in B. vulgaris ………………………………… 131.9 Objectives and scientific hypotheses ………………………………… 132.
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Aus dem Institut für Pflanzenbau und Pflanzenzüchtung
der Christian-Albrechts-Universität zu Kiel


Novel Genetic Factors Affecting Bolting and Floral Transition
Control in Beta vulgaris


Dissertation
zur Erlangung des Doktorgrades
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel

vorgelegt von
M.Sc. Salah Fatouh Abou-Elwafa
aus Sohag, Ägypten
Kiel, 2010

Dekan: Professor Dr. Karin Schwarz
1. Berichterstatter: Professor Dr. Christian Jung
2. Berichterstatter: Professor Dr. Daguang Cai
Tag der mündlichen Prüfung: 10.02.2010
Table of Contents I
Tables of Contents
Tables of Contents ………………………………………………………….……. I
Abbreviations ………………………………………………………………… V
List of Tables …………………………………………………………………. VII
List of Figures …………………………………………………………..……. VIII
1. General introduction ………………………………………...…………... 1
1.1 History of sugar beet cultivation…………………………………….. 1
1.2 Biology, taxonomy and breeding of sugar beet ……………………… 1
1.3 Sugar beet cultivation in non-European countries …………………… 6
1.4 Genetic analysis of sugar beet ……………………………………….. 7
1.5 The impact of early bolting on sugar beet production ……………….. 8
1.6 Environmental and internal factors regulating bolting and flowering .. 9
1.7 Flowering time regulation in model species …………………………. 10
1.8 Flowering time genes in B. vulgaris ………………………………… 13
1.9 Objectives and scientific hypotheses ………………………………… 13
2. Conservation and divergence of autonomous pathway genes in the
flowering regulatory network of Beta vulgaris: ………………….…..... 15
2.1 Abstract ……………………………………………………………… 15
2.2 Introduction………………………………………………………….. 15
2.3 Materials and Methods ………………………………………………. 20
2.3.1 Bioinformatic analyses …………………………………………. 20
2.3.2 Plant material and growth conditions …………………………... 21
2.3.3 BAC library screening ………………………………………….. 22
II Table of Contents
2.3.4 RT-PCR and RACE …………………………………………….. 22
2.3.5 Vector construction and transformation of A. thaliana ………… 23
2.3.6 Genetic mapping and statistical analysis ……………………….. 24
2.4 Results ……………………………………………………………….. 25
2.4.1 Beta vulgaris homologs of autonomous pathway genes ……….. 25
2.4.2 Genetic map positions ..…………………………………………. 28
2.4.3 BvFLK accelerates the time to bolting in Arabidopsis and
complements the flk1 mutation .………………………………. 29
2.4.4 BvFLK represses FLC expression in Arabidopsis ……………… 32
2.4.5 BvFVE1 does not complement an fve mutation in Arabidopsis … 32
2.4.6 Transcript accumulation of BvFVE1, but not BvFLK, is under
33
circadian clock control …………………………………………..
2.5 Discussion ……………………………………………………………. 35
2.6 Acknowledgements ………………………………………………….. 41
2.7 References …………………………………………………………… 41
3. A survey of EMS-induced biennial Beta vulgaris mutants reveals a
novel bolting locus which is unlinked to the bolting gene B…………… 52
3.1 Abstract ………………………………………………………………. 52
3.2 Introduction ………………………………………………………….. 52
3.3 Materials and Methods ………………………………………………. 57
3.3.1 Plant material …………………………………………………… 57
3.3.2 Phenotypic analysis ……………………………………………... 58
3.3.3 DNA extraction and genotypic analysis ……………………….. 59
Table of Contents III
3.3.4 Map construction and statistical analysis ……………………….. 60
3.4 Results ……………………………………………………………….. 60
3.4.1 Phenotypic segregation for annual bolting ……………………... 60
3.4.2 Co-segregation analysis of bolting phenotypes and B locus
marker genotypes: Evidence for additional bolting loci ………... 62
3.4.2.1 Co-segregation analysis in populations EW1, EW2 and
EW3 ……..…..……………………................................. 62
3.4.2.2 nalysis in populations EW4a and EW4b . 63
3.4.3 Variation in bolting time among annuals in F populations ……. 652
3.4.4 Genetic mapping of the bolting locus in population EW2 ……… 66
3.4.5 Co-segregation analysis of bolting behavior and the
chromosome IX marker MP_R0018 in populations EW1, EW3
and EW4a ……………..…..……………………...……………... 67
3.5 Discussion ……………………………………………………………. 68
3.6 Acknowledgements ………………………………………………….. 75
3.7 References …………………………………………………………… 75
4. Genetic mapping of a novel bolting locus (B4) linked to the B gene on
chromosome II of Beta vulgaris ………………………………………… 81
4.1 Abstract ………………………………………………………………. 81
4.2 Introduction ………………………………………………………….. 81
4.3 Materials and Methods ………………………………………………. 84
4.3.1 Plant material and phenotypic analysis ………………………… 84
4.3.2 Marker development and genetic mapping …………………….. 85
4.3.3 Statistical analysis ……………………………………………… 86
4.4 Results …………………………………………………….………… 86
IV Table of Contents

4.4.1 Phenotypic analysis of bolting behavior in F populations EW5a 2
86 and EW5b ……………………………………………………….
4.4.2 Co-segregation analysis of bolting phenotypes and B locus
marker genotypes ……………………………………………….. 88
4.4.3 Genetic mapping of B4 locus on chromosome II ………………. 89
4.4.4 Neither B nor B4 locus affects bolting time in populations EW5a
and EW5b ………………………………………………………. 91
4.5 Discussion ……………………………………………………………. 92
4.6 Acknowledgements ………………………………………………….. 95
4.7 References …………………………………………………………… 95
5. Closing Discussion ……………………………………………………….. 98
6. Summary …………………………………………………………………. 107
7. Zusammenfasung ……………………... 109
8. References ………………………………………………………………... 111
9. Supplementary data ……………………………………………………… 124
10. Acknowledgments …..………..…………………………………………... 125
11. Curriculum vitae …..……………..………………………………………. 126




Abbreviations V
Abbreviations
A Adenine
ABA Abscic acid
AFLP Amplified Fragment Length Polymorphism
ANOVA Analysis of Variance
BAC Bacterial artificial chromosome
bp Basepair(s)
BvCOL1 Beta vulgaris CONSTANS Like1
BvFL1 Flowering Locus1
C Cytosine
°C Degree celsius
CaMV 35S Cauliflower Mosaic Virus 35S Promoter
CAPS Cleaved amplified polymorphic sequence
cM Centimorgan
cm Centimeter
CMS Cytoplasmic male sterility
CO CONSTANS
Col-0 Arabidopsis Columbia background
DNA Deoxyribonucleic acid
dNTP 2`- deoxyribonucleotide- 5`- triphosphate
EMS Ethyl methanesulfonate
EST Expressed Sequence Tag
F First generation 1
F Second generation 2
F Third generation 3
FCA FLOWERING LOCUS CA
FLC Flowering Locus C
FLD FLOWERING LOCUS D
FLK Flowering Locus KH domains
FPA FLOWERING LOCUS PA
FT Flowering Locus T
FVE FLOWERING LOCUS VE
FY US Y
G Guanine
GA Gibberellins
h Hour
Ha Hectare
JA Jasmonic acid
kb Kilo base pairs = 1,000 bp
LD LUMINIDEPENDENS
VI Abbreviations
LOD Logarithm of odds
LSD Least significant difference
Mb Mega base pairs = 1,000,000 bp
Mg Milligram
min Minute
ml Milliliter
mM Millimolar
NASC Nottingham Arabidopsis Stock Centre
NCBI National Center for Biotechnology Information
ng Nanogram
nm Nanometer
PCR Polymerase Chain Reaction
qPCR Quantitative real-time polymerase chain reaction
QTL Quantitative Trait Loci
RACE Rapid amplification of complementary DNA ends
RAPD Random Amplified Polymorphic DNA
RFLP Restriction Fragment Length Polymorphisms
RNA Ribonucleic acid
RNAi RNA interference
RRM RNRecognition Motifs
RT-PCR Reverse transcription polymerase chain reaction
sec Second
SNP Single Nucleotide Polymorphism
SOC1 SUPPRESSOR of OVEREXPRESSION of CONSTANS 1
ssp. subspecies
SSR Simple Sequence Repeats
SVP SHORT VEGETATIVE PHASE
SWP1 SWIRM DOMAIN PAO PROTEIN 1
T Thymine
T-DNA Transfer DNA
UV Ultraviolet radiation
µg Microgram
µl Microliter
2 Chi square χ

List of Tables VII
List of Tables
Table 1: B. vulgaris ESTs with homology to A. thaliana autonomous pathway
genes ……………………………………………………………………………... 26
Table 2: Number of days to bolting (DTB) and total number of leaves at bolting
(TNL) of primary transformants (T1 generation) and transgenic plants in
segregating T2 populations derived from transformation of BvFLK and FLK into
A. thaliana Col-0 and the flk mutant SALK_112850 (flk1)……………………… 30
Table 3: Biennial EMS mutants and generation of F populations ……………... 582
Table 4: Phenotypic segregation for bolting behavior in F populations ……….. 622
Table 5: Co-segregation analysis of bolting behavior and B locus marker
genotypes ……..….………………………………………………………………. 64
Table 6: Analysis of variance among B locus marker genotypes in annual
subpopulations…...……………………………………………………………….. 66
Table 7: Co-segregation analysis of bolting behavior and chromosome IX
marker genotypes ………………………………………………………………. 68
Table 8: Analysis of variance among chromosome IX marker genotypes in
annual subpopulations …………………………………………………………… 69
Table 9: Phenotypic segregation for bolting behavior in F populations ……….. 872
Table 10: Co-segregation analysis of bolting behavior and the B locus marker
(GJ1001c16) genotypes in populations EW5a and EW5b ….…………………… 88
Table 11: Analysis of variance and T-test among B and B4 locus marker
genotypes in annual subpopulations ……………….…………………………….. 91
VIII List of Figures
List of Figures
Figure 1: Sequence and structure of the autonomous pathway gene homologs
BvFLK and BvFVE1 .………………………………………………………….. 27
Figure 2: Genetic map positions ...……………………………………………. 28
Figure 3: complementation analysis of BvFLK in the A. thaliana flk1 mutant.. 31
Figure 4: Expression of BvFLK and BvFVE1 in B. vulgaris ……………...….. 34
Figure 5: Test for allelism between EMS mutations and the B locus ………... 56
Figure 6: Phenotypic segregation for bolting behavior in F populations..…... 622
Figure 7: Genetic map position of a major locus for annual bolting on
chromosome IX ……………………………………………………………….. 67
Figure 8: Phenotypic segregation for bolting behavior in F populations ...…. 872
Figure 9: Allelism test between EMS-induced mutation and the B locus ....… 89
Figure 10: Genetic map position of B4 locus for annual bolting on
chromosome II …..…………………………………………………………….. 90
Figure 11: Genetic map positions of bolting loci and floral transition genes in
sugar beet in mapping populations ...…………………………….……………. 101


General introduction 1
1. General introduction
1.1. History of sugar beet cultivation
Sugar beet (Beta vulgaris ssp. vulgaris L.) is a herbaceous dicotyledon. The
cultivated form is biennial. It grows vegetatively in its first year as a near-rosette
plant and develops a large fleshy taproot that contains the food reserve for the
second year of growth. In the second year, sugar beet becomes reproductive
(Biancardi 2005).
Beets grown as vegetables are shown in 4000-year old Egyptian temple artwork;
however, their use as a sugar crop is relatively recent. Beet had been known as a
vegetable crop in different civilizations in the Mediterranean region, including
Egyptian, Greek and Roman. It had been grown mainly for leaves, and it might be
similar to what is known today as Swiss chard. Many names for beet in different
ancient languages (e.g. selg in Arabic and silg in Nabataean) are apparently derived
from the Greek word sicula used by Theophrastus ca. 300 B.C. (Biancardi
2005;McGrath et al., 2007). More detailed accounts of the history of sugar beet are
given by Biancardi 2005.
Sugar beet was cultivated regularly in the field in the seventeenth century, therefore
it is much newer than other traditional crops. As early as in the sixteenth century,
the French botanist Olivier de Serres extracted a sweet syrup from beet roots. The
Prussian chemist Andreas Sigismund Marggraf used alcohol to extract sugar from
beets in 1747. Because the sugar content of the roots of Beta which he investigated
was very low (1.6% of the root fresh weight), this method did not lend itself to
industrial scale production (Poggi 1980).
Breeding of sugar beet started in the beginning of the nineteenth century by Franz
Carl Achard, a former student and successor of Marggraf, who obtained by mass
selection the variety White Silesian which had 5-7% sugar content (Bosemark
1993). By the middle of the nineteenth century the first sugar beet variety with a
relatively high-sugar content (~14%), “Imperial Rübe”, had been released. By the
beginning of the twentieth century and with the discoveries of Mendelian genetics
and Fischer’s statistical analysis, breeding of sugar beet was proceeding much
faster (Bosemark 1993).
1.2. Biology, taxonomy and breeding of sugar beet
Sugar beet is a biennial species germinating epigeally and forming a rosette of
glossy dark green leaves with outstanding petioles and conical storage roots in the
first year. The reproductive development by which the plant morphology becomes

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