modification of fatty acid composition in soybean seeds to improve soybean oil quality and ...

228 Pages·2011·2.32 MB·English

by  Anh Tung Pham

MODIFICATION OF FATTY ACID COMPOSITION IN SOYBEAN SEEDS TO IMPROVE
SOYBEAN OIL QUALITY AND FUNCTIONALITY

A Dissertation
Presented to
The Faculty of the Graduate School
At the University of Missouri

In Partial Fulfillment
Of the Requirements for the Degree
DOCTOR OF PHILOSOPHY

By
Anh TUNG PHAM
Dr. Kristin Bilyeu, Dissertation Supervisor

July 2011
The undersigned, appointed by the dean of the Graduate School,
have examined the Dissertation entitled
MODIFICATION OF FATTY ACID COMPOSITION IN SOYBEAN SEEDS TO IMPROVE
SOYBEAN OIL QUALITY AND FUNCTIONALITY
Presented by Anh Tung Pham
A candidate for the degree of
DOCTOR OF PHILOSOPHY OF PLANT BIOLOGY AND GENETICS
And hereby certify that, in their opinion, it is worthy of acceptance.

Dr. Kristin D. Bilyeu

Dr. David A. Sleper

Dr. J. Grover Shannon

Dr. Emmanual Liscum
ACKNOWLEDGEMENTS

I would like to express my gratitude to Dr. Kristin Bilyeu not only for giving me
the opportunity to join her research group and conduct exciting research in plant biology
and genetics, but also for her continuous advising, support, and encouragement during
my doctoral studies. The quality of this study was greatly influenced by her help in
avoiding adversities and in solving problems which arose during the course of the
research project, and also by her research ethics and her perseverance for a deeper
understanding of this project.
I would also like to thank Dr. Grover Shannon for his collaboration and
contribution to my dissertation projects. I would like to thank Dr. David Sleper for your
guidance and support. I would like to thank Dr. David Sleper, Dr. Grover Shannon, Dr.
Mannie Liscum for serving on my doctoral committee. Also, I would like to thank all the
professors from different educational levels who have encouraged me or inspired me, one
way or another, to appreciate academia and to pursue an advanced degree.
I greatly acknowledge the help of the current and past members of Dr.Shannon‟s
group and Dr. Bilyeu‟s group: Dr. Jeong-Dong Lee for his helpful insights and for
supplying the germplasm and segregating populations used in this study, Dr. Jason
Gillman who share with me your deep knowledge and your willingness to help with all
the difficulties that I encountered during the course of the study. My thanks also are for
Kerry Clark, Christi Shannon, Stewart Selves and Paul Little for helping me with field
works, protein, oil and fatty acid analysis. Thanks and good luck to my colleagues Ashley
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Tetlow, Katherine Hagely, and Julian Lenis. You have created a family-like asmosphere
that makes me enjoy and appreciate our friendship.
At last, many thanks to all friends that I was fortunate to meet in Columbia MO
and had so much fun with. I am so happy that we shared a part of our “journeys” and I
really hope to stay in touch. I am especially thankful to all you that our friendship has
already lasted so long and your support has not changed, although so far away. I am
also grateful to my family, especially my parents, Binh Nguyen and Minh Pham, and my
brother, Linh Pham, for their love and understanding. I deeply wish that in the near future
I will be able to share more time with you. Finally, I would like to thank
my husband, Giang Nguyen for being there to share my excitement and disappointment,
encourage me and keep my motivation at all times, and make me smile even those days
that “everything went wrong”

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................................ ii
CHAPTER 1 Literature review .......................................................................................... 1
Figure ....................................................................................................................... 49
Literature cited ........................................................................................................ 51
CHAPTER 2 Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans
with the high oleic acid seed oil trait ................................................................................ 69
Abtract ...................................................................................................................... 71
Introduction ............................................................................................................. 72
Materials and methods ............................................................................................ 77
Population development......................................................................... 77
DNA isolation and PCR for sequencing of FAD2-1A and FAD2-1B .... 79
Sequence analysis .................................................................................. 79
FAD2-1B allele specific molecular marker assay ................................. 80
FAD2-1A allele specific molecular marker assay for 17D .................... 81
FAD2-1A allele specific molecular marker assay for M23 ................... 81
Fatty acid, protein and oil determination ............................................... 82
Population genotyping ........................................................................... 83
Results ..................................................................................................................... 84
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Identification of mutant alleles of FAD2-1B in soybean lines PI 283327
and PI 567189A ..................................................................................... 84
The PI 283327 FAD2-1B allele is associated with an increase in seed
oleic acid content ................................................................................... 86
Combinations of mutations in FAD2-1A and FAD2-1B produce high
oleic acid levels in the seed oil .............................................................. 87
Excess desaturase activity: A single wild-type FAD2-1 allele prevents
high oleic acid accumulation ................................................................. 90
The high oleic acid phenotype is stable in plants grown in alternate
environments .......................................................................................... 91
Full fatty acid profiles and total oil and protein content ........................ 92
Discussion ............................................................................................................... 94
Figures ................................................................................................................... 100
Tables .................................................................................................................... 108
Literature cited ...................................................................................................... 111
CHAPTER 3 A novel FAD2-1A allele in a soybean plant introduction offers an alternate
means to produce soybean seed oil with 85% oleic acid content ................................... 115
Summary ............................................................................................................... 116
Introduction ........................................................................................................... 117
Materials and method ............................................................................................ 121
DNA isolation and sequencing of FAD2-1A and FAD2-1B ................ 121
Population development....................................................................... 121
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FAD2-1A allele specific molecular marker assay ................................ 122
Fatty acid, protein and oil determination ............................................. 123
Population genotyping using SimpleProbe assay ................................ 123
Design of field experiments ................................................................. 124
Statistical analysis ................................................................................ 125
Results ................................................................................................................... 126
Identification of novel alleles of FAD2-1A and FAD2-1B in soybean
plant introductions ............................................................................... 126
Combinations of novel mutant alleles of FAD2-1A with mutant FAD2-
1B alleles produce high oleic acid levels in the seed oil ...................... 128
Full fatty acid profiles and total oil and protein content ...................... 130
Discussion ............................................................................................................. 133
Figures ................................................................................................................... 137
Tables .................................................................................................................... 139
Liturature cited ...................................................................................................... 145
CHAPTER 4 Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid
and low linolenic acid soybean oil .................................................................................. 148
Summary ............................................................................................................... 149
Introduction ........................................................................................................... 150
Materials and methods .......................................................................................... 155
Population development....................................................................... 155
FAD2-1A allele specific molecular marker assay ................................ 157
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Genotyping for the gene combination of interest ................................ 158
Fatty acid determination ...................................................................... 158
Stability of high oleic/low linolenic soybean....................................... 159
Statistical analysis ................................................................................ 160
Results ................................................................................................................... 161
Maturity of the HOLL lines ................................................................. 162
For the HOLL soybean lines with null FAD2-1A(∆) alleles derived from
M23 ...................................................................................................... 162
For the HOLL soybean lines with S117N missense FAD2-1A alleles
from 17D .............................................................................................. 165
Agronomic characteristics of HOLL lines ........................................... 167
Oil and protein content......................................................................... 167
Discussion ............................................................................................................. 169
Figures ................................................................................................................... 175
Tables .................................................................................................................... 178
Liturature cited ...................................................................................................... 182
CHAPTER 5 Identification of candidate genes encoding for three acyltransferase
enzymes controlling the triacylglycerol biosynthesis in soybean seed ........................... 186
Summary ............................................................................................................... 187
Introduction ........................................................................................................... 188
Materials and methods .......................................................................................... 193
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Database search for Arabidopsis sequences of GPAT, LPAAT and DGAT
genes ....................................................................................................... 193
Homologous sequence identification and transcriptional expression data
mining ..................................................................................................... 193
RNA isolation, reverse transcriptase reactions, and quantitative Real-Time
PCR ......................................................................................................... 194
Results ................................................................................................................... 195
Discussion ............................................................................................................. 199
Figures ................................................................................................................... 203
Tables .................................................................................................................... 208
Liturature cited ...................................................................................................... 211
VITA ............................................................................................................................... 216

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LIST OF ILLUSTRATION
LIST OF FIGURES
Figure Page
1.1 Generalized scheme for triacylglycerol (TAG) assembly in developing
seeds of oleaginous plants ........................................................................... 50
2.1 Characterization of mutations in the FAD2-1B alleles from soybean lines
PI 283327 and PI 567189A ....................................................................... 101
2.2 Seed oleic acid phenotype and FAD2-1 genotype association analysis for
population 1 .............................................................................................. 103
2.3 Seed oleic acid phenotype and FAD2-1 genotype association analysis for
population 2 .............................................................................................. 104
2.4 Seed oleic acid phenotype and FAD2-1 genotype association analysis for
population 3 .............................................................................................. 105
2.5 Seed oleic acid phenotype and FAD2-1 genotype association analysis for
population 4 .............................................................................................. 106
2.6 Seed oleic acid phenotype and FAD2-1 genotype association analysis for
F seeds of population 4 ............................................................................ 107
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3.1 Weblogo output of the amino acid conservation FAD2 enzyme as part of
the BLINK feature at NCBI using GI number 197111722 ....................... 138
4.1 Seed linoleic (18:2) and linolenic acid (18:3) contents of high oleic low
linolenic acid (HOLL) soybean lines developed with null allele ofM23
FAD2-1A in Portageville Missouri summer 2010 .................................... 176
4.2 Seed linoleic (18:2) and linolenic acid (18:3) content of HOLL soybean
lines developed with either M23 or 17D FAD2-1A alleles in Columbia
summer 2010 ............................................................................................. 177

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