Evaluation of agronomic, seed quality traits, and transcript abundance in conventional high oleic

85 Pages·2017·0.92 MB·English

by  Lauren Kathryn Richardson

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Masters Theses Graduate School
5-2016
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Lauren Kathryn Richardson
University of Tennessee - Knoxville, [email protected]
Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes
Part of the Agriculture Commons
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Richardson, Lauren Kathryn, "Evaluation of agronomic, seed quality traits, and transcript abundance in
conventional high oleic soybeans with mutant FAD2-1A and FAD2-1B genes. " Master's Thesis, University
of Tennessee, 2016.
https://trace.tennessee.edu/utk_gradthes/3800
This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and
Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE:
Tennessee Research and Creative Exchange. For more information, please contact [email protected].
To the Graduate Council:
I am submitting herewith a thesis written by Lauren Kathryn Richardson entitled "Evaluation of
agronomic, seed quality traits, and transcript abundance in conventional high oleic soybeans
with mutant FAD2-1A and FAD2-1B genes." I have examined the final electronic copy of this
thesis for form and content and recommend that it be accepted in partial fulfillment of the
requirements for the degree of Master of Science, with a major in Plant Sciences.
Vincent R. Pantalone, Major Professor
We have read this thesis and recommend its acceptance:
Hem Bhandari, Feng Chen, Tarek Hewezi
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
Evaluation of agronomic, seed quality traits, and transcript
abundance in conventional high oleic soybeans with mutant
FAD2-1A and FAD2-1B genes

A Thesis Presented for the
Master of Science
Degree
The University of Tennessee, Knoxville

Lauren Kathryn Richardson
May 2016
Copyright © 2016 by Lauren K. Richardson
All rights reserved.

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ACKNOWLEDGEMENTS
The completion of this thesis project and graduate program would not
have been possible without the constant encouragement and guidance of others.
I would like to thank my advising professor, Dr. Vince Pantalone, for giving me
the incredible opportunity to see into the world of plant breeding despite having
no previous experience. Dr. Pantalone has allowed me to not only learn in the
classroom, but to learn through hands on experience in the field and through
numerous trips to professional meetings and workshops. He has a particular gift
for enabling his students to succeed by showing confidence in their abilities and
through his care for professional and personal well-being. I would also like to
thank Dr. Tarek Hewezi for allowing me to work in his lab to gain new experience
in molecular biology. I would also like to acknowledge Dr. Hem Bhandari and Dr.
Feng Chen for serving as advising committee members and exceptional
teachers.
Next, I would like to thank all members of the ‘Bean Team’ for helping me
with this project every step of the way and for making graduate school such a
positive experience. I would like to acknowledge Chris Smallwood and Rachel
Fulton for their never ending supply of pep-talks and great patience. Also, I would
like to thank the farm crew and fellow graduate students for being constant
sources of help and encouragement.
Finally, I would like to thank my family, dear friends, and fiancé for being a
never ending supply of love and support over the past two years.
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ABSTRACT
Two soybean omega-6 fatty acid desaturase genes [FAD2-1A and FAD2-
1B] are responsible for converting oleic acid into linoleic acid (Okuley et al.,
1994). Plant introductions [PI] 603452 and 283327 have naturally occurring
mutations in FAD2-1A and FAD2-1B, respectively, which hinder the conversion
of oleic acid to polyunsaturated fatty acids. The end result is more functional
soybean oil with exceptionally high levels of oleic acid. The homozygous alleles
of FAD2-1A and FAD2-1B were combined in a BC F [backcross three, second
3 2:4
filial generation selected, advanced to the fourth filial generation] population of
forty-eight lines and were evaluated in Tennessee. Each line belonged to a
double homozygous genotypic class: i) double wild-type [WT] (AABB), ii) mutant
FAD2-1A with WT FAD2-1B (aaBB), iii) the converse (AAbb), and iv) double
mutant (aabb). All genotypic groups yielded similarly to each other and to three
high yielding checks (p<0.05). When one mutant allele was present the high oleic
content was increased significantly (aaBB-34.4% and AAbb-26.2%) compared to
AABB-21.3%. When both mutations were combined, aabb, the mean oleic
content was significantly greater (p<0.05) than all other genotypic groups
(80.4%). The range of oleic acid content across all twelve double mutant groups
was 77.1% - 81.8%. It appears that the high oleic genotype has a significant
effect on total oil, crude protein, and the concentration of five amino acids. The
aabb group had significantly more oil (240 ± 1 g kg-1) and crude protein (400 ± 7
g kg-1) than all other groups. This increase in crude protein is consistent with the
significantly lowered amino acid concentration (p<0.05) of lysine, methionine,
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tryptophan, and threonine in the aabb group. Conversely, the high oleic group
had the greatest concentration of cysteine (p<0.05). It appears that mutant
FAD2-1 genes from PI 603452 and PI 283327 are a useful source for developing
high oleic soybean lines in Tennessee with no yield drag. It will be beneficial to
explore the effect of these mutant alleles on total oil and protein in the future.

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TABLE OF CONTENTS

INTRODUCTION .................................................................................................. 1
Breeding High Oleic Soybeans.......................................................................... 3
Objectives ...................................................................................................... 7
References ........................................................................................................ 8

CHAPTER I Evaluation of agronomic traits of 48 BC F derived lines belonging
3 2:4
to four double homozygous genotypic classes for the FAD2-1A and FAD2-1B loci
............................................................................................................................ 13
Abstract ........................................................................................................... 14
Introduction ..................................................................................................... 15
Materials and Methods .................................................................................... 15
Plant Materials ............................................................................................. 15
DNA Extraction and SNP Genotyping .......................................................... 19
Agronomic Traits .......................................................................................... 20
Data Analysis ............................................................................................... 21
Results and Discussion ................................................................................... 22
References ...................................................................................................... 25
Appendix ......................................................................................................... 27

CHAPTER II Evaluation of concentration and quality of seed oil and protein of 48
field tested BC3F2:4 soybeanLines ....................................................................... 33
Abstract ........................................................................................................... 34
Introduction ..................................................................................................... 34
Materials and Methods .................................................................................... 37
Gas Chromatography .................................................................................. 37
Near Infrared Reflectance ............................................................................ 38
Results and Discussion ................................................................................... 38
Fatty Acid Concentrations ............................................................................ 38
Oil, Protein, and Amino Acids ...................................................................... 39
References ...................................................................................................... 43
Appendix ......................................................................................................... 46

CHAPTER III Evaluation of transcript abundance of FAD2-1A and FAD2-1B with
different allelic combinations at different growth stages of soybean ................... 56
Abstract ........................................................................................................... 57
Introduction ..................................................................................................... 57
Materials and Methods .................................................................................... 59
Plant Growth and Sample Collection ........................................................... 59
RNA Extraction and Sample Preparation ..................................................... 60
Reverse Transcription Quantitative PCR ..................................................... 62
Results and Discussion ................................................................................... 64
References ...................................................................................................... 68
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Appendix ......................................................................................................... 70

CONCLUSION .................................................................................................... 72
References ...................................................................................................... 73
VITA .................................................................................................................... 74

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LIST OF TABLES

Table 1.1 Yield (kg ha-1), maturity (DAP), height (cm), and lodging of 48
experimental lines, three high yielding checks, and recurrent parent,
TN10-4037, averaged over three replications and three locations ....
...................................................................................................... 31
Table 2.1 Fatty acid mean concentrations of 12 BC F lines in each of four
3 2:4
experimental groups with different FAD2-1A and FAD2-1B genotypes
...................................................................................................... 46
Table 2.2 Protein, oil, and amino acid concentration means of 12 BC F lines in
3 2:4
each of four experimental groups with different FAD2-1A and FAD2-1B
genotypes4 ...................................................................................... 8
Table 2.3 Oil, protein, and fatty acid concentrations of 48 experimental lines,
three high yielding checks, and recurrent parent, TN10-4037, averaged
over three replications and three locations .................................... 49
Table 2.4 Ratio of mean grams of amino acids per mean grams of crude protein
of 48 experimental lines, three high yielding checks, and recurrent parent,
TN10-4037, averaged over three replications and three locations ....
...................................................................................................... 53
Table 3.1 FAD2-1A and FAD2-1B genotype for experimental and control lines .....
.......................................................................................................... 70
Table 3.2 FAD2-1A mRNA fold change relative to FAD2-1B ................... 70

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