Genetic Basis of Tocopherol Accumulation in Soybean

247 Pages·2012·3.42 MB·English

Genetic Basis of Tocopherol Accumulation in
Soybean (Glycine Max [L.] Merr.) Seeds

by

Eric Jonathan Shaw

A Thesis
presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Plant Agriculture

Guelph, Ontario, Canada

© Eric J. Shaw, September, 2012
ABSTRACT

GENETIC BASIS OF TOCOPHEROL ACCUMULATION IN SOYBEAN (GLYCINE
MAX [L.] MERR.) SEEDS

Eric Shaw Advisor:
University of Guelph, 2012 Dr. Istvan Rajcan

This thesis is an investigation of the genetic basis of tocopherol accumulation in soybean
(Glycine max [L.] Merrill) seeds. Soybean is the world’s most widely grown protein and oilseed
crop and the principle source of vitamin E (tocopherols) as a supplement. Tocopherols (α-, β-, γ-
and δ-isomers) are powerful antioxidants that contain human health benefits, including a
decrease in the risk of lung cancer, heart disease and osteoporosis. The purpose of this research
was to identify genetic and biochemical components affecting tocopherol accumulation in
soybean seeds. The objectives were to: 1) investigate location and year effects on soybean seed
tocopherol levels in the field; 2) determine environmental factors affecting soybean seed
tocopherol levels under controlled conditions; 3) identify simple sequence repeat (SSR) markers
that tag quantitative trail loci (QTL) for individual and total tocopherols; and 4) evaluate the
potential role of VTE1, VTE3 and VTE4 genes in tocopherol accumulation using the candidate
gene approach. Seventy nine recombinant inbred lines (RILs) derived from the cross between
OAC Bayfield and OAC Shire were grown in the field at Elora, Woodstock and St. Pauls, ON, in
2009 and 2010. The tocopherol components were quantified using high performance liquid
chromatography (HPLC). The results showed a significant (p < 0.001) genotype, environment
and genotype x environment effect for each tocopherol component. It was discovered that a 2 x
phosphate fertilizer (K SO at 1.0M/150mL) and 30 ˚C temperature treatment increased each
2 4
tocopherol component, whereas drought had no effects. Single marker analysis identified 42
QTL and interval mapping identified 26 QTL across 17 chromosomes. Significant two-locus
epistatic interactions were found with a total of 122 and 152 in the 2009 and 2010 field seasons,
respectively. The multiple locus models explained 18.4% to 72.2% with an average of 45.7% of
the total phenotypic variation. The candidate gene approach using nucleotide sequences from the
coding regions identified two single nucleotide polymorphisms (SNPs) in VTE1, five SNPs in
VTE4 and none in VTE3. The SNPs were predicted to cause functional protein changes and the
genes co-localized with some of the identified QTL. The results of this study provide a better
understanding of the environmental factors and genetic mechanisms that influence the
accumulation of tocopherols in soybean seeds.
Acknowledgements

I would like to express my extreme gratitude to everyone who has supported me during
my research. Firstly, thank you to my advisor Dr. Istvan Rajcan for being a superb advisor and
great friend during the process. I have learned numerous new research and development skills
throughout the years at the University of Guelph and Dr. Rajcan’s expertise and attitude towards
research greatly enhanced my experience.
Secondly, I wish to thank Dr. Yukio Kakuda, Dr. Ian Tetlow and Dr. Ali Navabi for
serving on my advisory committee. They provided me with some great advice over the course of
my thesis. I would like to extend the thank you to Dr. Kakuda for providing help with the HPLC
and for providing the use of the equipment in his lab. Thank you to Dr. Duane Falk, Dr. Ali
Navabi and Dr. Istvan Rajcan for serving on the examination committee, to Dr. Barry Shelp for
serving as the chair and to Dr. Steven Britz for serving as my external examiner.
Thirdly, thank you to the soybean lab including Wade Montminy, Alberto Aguilera,
Yesenia Salazar, Tim Currie, Lin Liao, Martha Lucia and Mei Wu and others in the program for
their help in 2009 and 2010. Of course, thank you to Chris Grainger for all of his help in the
molecular lab.
Special thanks to my dad, brother, and sister for their support and to my entire family for
giving me great direction and advice over the years. Thanks to all my friends as well.
Finally, thank you to Terri for all of her continued love and support throughout the years.
Our two dogs, Troy and Sparta deserve recognition for all of their antics and entertainment over
the years.

iv
List of Abbreviations

ANOVA Analysis of variance
α-TTP α-tocopherol transfer protein
CHUs Crop heat units
CV Coefficient of variation
DF Degrees of freedom
DMPBQ 2,3-dimethyl-6-phytyl-1,4-benzoquinone
dNTP Deoxyribo-nucleotide triphosphate
EtOH Ethyl alcohol
ESTs Expressed sequence tags
F-Value F test statistics
HGA Homogentisic acid
HPBQ MT 2-methyl-6-phytylbenzoquinol methyltransferase
HPLC High performance liquid chromatography
HPPD p-hydroxy-phenylpyruvate dioxygenase
HPT Homogentisate phytyltransferase
IU International units
K Potassium
KOH Potassium hydroxide
LG Linkage groups
LOD Likelihood of odds
MAS Marker assisted selection
MPP Methylerythritol phosphate pathway
ML Monte Carlo maximum likelihood
NCBI National Center for Biotechnology Information
NIR Near infrared reflectance spectrometry
NN Nearest neighbour
P Phosphorus
PCR Polymerase chain reaction
PCT Phosphatidate cytidylyltransferase
QTL Quantitative trait loci
RCBD Randomized complete block design
RFLP Restriction fragment length polymorphism
ROS Reactive oxygen species
RILs Recombinant inbred lines
R² Coefficient of determination
SCL Strongest cross link
SD Standard deviation
SE Standard error
SNP Single nucleotide polymorphism
SP Shikimate pathway
SSR Simple sequence repeats
TC Tocopherol cyclase
γ-TMT γ-tocopherol methyltransferase
v
Table of Contents
Acknowledgement iv

List of Abbreviations v

Table of Contents vi

List of Tables ix

List of Figures xiv

Appendices
List of Tables xvii
List of Figures xviii

General Introduction 1

Chapter One - Literature Review 5

1.1 Soybean History 6
1.2 Vitamin E Discovery 7
1.3 Tocopherol Chemical Structure 8
1.4 Tocopherol in Soybean Seeds 9
1.5 Tocopherol Content Heritability in Soybean Seeds 11
1.6 Tocopherol Utilization in the Human Body 12
1.7 The Tocopherol Biosynthetic Pathway 13
1.8 Human Health Benefits of Vitamin E 16
1.9 Molecular Markers 17
1.10 Soybean Genome 19
1.11 Concluding Remark 19

Chapter Two - Tocopherol Accumulation in Soybean Seed Produced Under
Various Field Environments 29

2.1 Abstract 30
2.2 Introduction 32
2.3 Materials and Methods 34
2.3.1 Plant Material for Field Studies 34
2.3.2 Agronomic Data Collection 35
2.3.3 Tocopherol Analysis 36
vi
2.3.4 Statistical Analysis 39
2.4 Results 41
2.4.1 Environmental Factors Affecting Tocopherols Accumulation under Field
Conditions 41
2.4.2 Relationship between Agronomic Traits and Tocopherols 44

2.5 Discussion 46

Chapter Three - Tocopherol Accumulation in Soybean Seed Produced Under
Controlled Environmental Conditions 69

3.1 Abstract 70
3.2 Introduction 71
3.3 Materials and Methods 74
3.3.1 Plant Material for Growth Room Studies 74
3.3.2 Fertilizer Treatment Growing Conditions 75
3.3.3 Temperature Treatment Growing Conditions 75
3.3.4 Drought Treatment Growing Conditions 76
3.3.5 Experimental Setup 76
3.3.6 Tocopherol Analysis 77
3.3.7 Statistical Analysis 77
3.4 Results 78
3.4.1 Fertilizer Treatment 78
3.4.2 Temperature Treatment 79
3.4.3 Drought Treatment 80
3.5 Discussion 82
3.5.1 Fertilizer Treatment 82
3.5.2 Temperature Treatment 83
3.5.3 Drought Treatment 85

Chapter Four - Molecular Mapping of Soybean Seed Tocopherols 93

4.1 Abstract 94
4.2 Introduction 96
4.3 Materials and Methods 99
4.3.1 Plant Material 99
4.3.2 Tocopherol Analysis 99
4.3.3 DNA Extraction 99
4.3.4 Molecular Marker Analysis 101
4.3.5 Data Analysis 103
4.3.5.1 Two-Locus Epistatic Interactions 104
4.3.5.2 Multiple Locus Model 104
4.4 Results 105
4.4.1 Linkage Map Construction 105
4.4.2 Molecular Marker Analysis 106
4.4.3 Interval Mapping 108
vii
4.4.4 Two-Locus Epistatic Interactions 110
4.4.5 Multiple Locus Model 111
4.5 Discussion 112

Chapter Five – Molecular Analysis of the Candidate Genes Involved in
Tocopherol Accumulation 132

5.1 Abstract 133
5.2 Introduction 135
5.3 Materials and Methods 138
5.3.1 Plant Material 138
5.3.2 DNA Extraction 138
5.3.3 Primer Design and PCR Optimization 138
5.3.4 Gene Sequencing 140
5.3.5 Data Analysis 142
5.4 Result 144
5.4.1 VTE1 - Tocopherol cyclase 144
5.4.2 VTE3 - 2-methyl-6-phytylbenzoquinol methyltransferase 146
5.4.3 VTE4 - γ-tocopherol methyltransferase 146
5.5 Discussion 150
5.5.1 VTE1 - Tocopherol cyclase 151
5.5.2 VTE3 - 2-methyl-6-phytylbenzoquinol methyltransferase 153
5.5.3 VTE4 - γ-tocopherol methyltransferase 155

General Discussion 188

References 194

Appendix Tables 202

Appendix Figures 212

viii
List of Tables

1. Table 1.1 - Natural food sources for Vitamin E (US. Department of Agriculture, 2004).21
2. Table 1.2 - Daily recommended dosage of Vitamin E based on gender and age (Hillan,
2008)..................................................................................................................................22
3. Table 2.1 – Average total tocopherol levels in the 79 soybean RILs derived from the
cross between OAC Bayfield x OAC Shire and checks for the 2009 and 2010 field
seasons located at the Elora, Woodstock and St.Pauls crop research stations...................50

4. Table 2.2 - Tocopherol values for α-, γ-, δ-, and total tocopherol for the RIL population
and parents at Elora, Woodstock and St.Pauls field locations in 2009..............................52

5. Table 2.3 - Tocopherol values for α-, γ-, δ-, and total tocopherol for the RIL population
and parents at Elora, Woodstock and St.Pauls field locations in 2010..............................53

6. Table 2.4 - Combined analysis of variance on α-tocopherol showing significant effects
on the RIL population, at Elora, Woodstock and St. Pauls in 2009 and
2010....................................................................................................................................54

7. Table 2.5 - Combined analysis of variance on γ- tocopherol showing significant effects
on the RIL population, at Elora, Woodstock and St. Pauls in 2009 and
2010....................................................................................................................................55

8. Table 2.6 - Combined analysis of variance on δ- tocopherol showing significant effects
on the RIL population, at Elora, Woodstock and St. Pauls in 2009 and
2010....................................................................................................................................56

9. Table 2.7 - Combined analysis of variance on total tocopherol showing significant effects
on the RIL population, at Elora, Woodstock and St. Pauls in 2009 and 2010...................57

10. Table 2.8 – Pearson Correlation Coefficients for α-, γ-, δ-, and total tocopherols for 8
phenotypic agronomic parameters using the RIL population grown at Elora, St. Pauls,
and Woodstock, Ontario in 2009.......................................................................................58

11. Table 2.9 – Pearson Correlation Coefficients for α-, γ-, δ-, and total tocopherols for 8
phenotypic agronomic parameters using the RIL population grown at Elora, St. Pauls,
and Woodstock, Ontario in 2010.......................................................................................59

12. Table 2.10 – Pearson correlation coefficients for α-, γ-, δ-, and total tocopherols using the
RIL population grown at Elora, St. Pauls, and Woodstock, Ontario in
2009....................................................................................................................................60

ix
13. Table 2.11 – Pearson correlation coefficients for α-, γ-, δ-, and total tocopherols using the
RIL population grown at Elora, St. Pauls, and Woodstock, Ontario in
2010....................................................................................................................................61

14. Table 2.12 – Combined Pearson correlation coefficients for the 8 phenotypic agronomic
parameters using the RIL population grown at Elora, St. Pauls, and Woodstock, Ontario
in 2009...............................................................................................................................62

15. Table 2.13 – Combined Pearson correlation coefficients for the 8 phenotypic agronomic
parameters using the RIL population grown at Elora, St. Pauls, and Woodstock, Ontario
in 2010...............................................................................................................................63

16. Table 2.14 – Variance partitioning and broad-sense heritability estimates for α-, γ- and δ-
tocopherols using the 79 RILs derived from cross OAC Bayfield x OAC Shire combined
over years and locations.....................................................................................................64

17. Table 3.1 - Mean tocopherol values for α-, γ-, δ-, and total tocopherol components for
RILs under standard, 2 x potassium and 2 x phosphate fertilizer treatments....................87

18. Table 3.2 - Mean tocopherol values for α-, γ-, δ-, and total tocopherol components for
high, medium and low tocopherol RILs and parents in response to standard, 2 x
potassium and 2 x phosphorous fertilizer treatments.........................................................88

19. Table 3.3 – Mean tocopherol values for α-, γ-, δ-, and total tocopherol components for
RILs grown under 30 ˚C, 25 ˚C and 20 ˚C temperature treatments...................................89

20. Table 3.4 - Mean tocopherol values for α-, γ-, δ-, and total tocopherol components for
RILs including patents in response to 30 ˚C, 25 ˚C and 20 ˚C temperature treatments....90

21. Table 3.5 – Mean tocopherol values for α-, γ-, δ-, and total tocopherol components for
RILs grown under 100%, 75% and 50% field capacity drought treatments......................91

22. Table 3.6 - Mean tocopherol values for α-, γ-, δ-, and total tocopherol components for
RILs including parents in response to 100%, 75% and 50% field capacity drought
treatments...........................................................................................................................92

23. Table 4.1 - QTL significantly associated with α-, γ-, δ-, and total soybean seed
tocopherol concentration using the RIL population derived from the original cross
between OAC Bayfield x OAC Shire at the Elora, Woodstock and St.Pauls crop research
stations in 2009 and 2010................................................................................................119
24. Table 4.2 – QTL significantly associated with more than one tocopherol components in
Elora, Woodstock and St. Pauls field locations using the RIL population derived from the
OAC Bayfield x OAC Shire cross in 2009 and 2010......................................................122

x