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12-2018 Generation and Evaluation of Modified opaque-2 (o2) Popcorn Suggests a Route to Quality Protein Popcorn (QPP) Ying Ren University of Nebraska-Lincoln
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This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses, Dissertations, and Student Research in Agronomy and Horticulture by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. GENERATION AND EVALUATION OF MODIFIED OPAQUE-2 (o2) POPCORN
SUGGESTS A ROUTE TO QUALITY PROTEIN POPCORN (QPP)
by
Ying Ren
A DISSERTATION
Presented to the Faculty of
The Graduate College at the University of Nebraska
In Partial Fulfillment of Requirements
For the Degree of Doctor of Philosophy
Major: Agronomy and Horticulture
(Plant Breeding and Genetics)
Under the Supervision of Professor David R. Holding
Lincoln, Nebraska
December, 2018 GENERATION AND EVALUATION OF MODIFIED OPAQUE-2 (o2) POPCORN SUGGESTS A ROUTE TO QUALITY PROTEIN POPCORN (QPP) Ying Ren, Ph.D. University of Nebraska, 2018
Advisor: David R. Holding
I have been working on a Quality Protein Popcorn breeding project where QPM conversion is carried out simultaneously for several elite popcorn germplasms. During my study in the graduate program, I led the following aspects of the Quality Protein Popcorn Breeding
Project:
1. Identified suitable QPMs as opaque-2 allele donors.
2. Examined the feasibility of quick introgression of the opaque-2 allele into
popcorn lines via marker-assisted selection.
3. Monitored modification by SDS-PAGE zein profiling and light box phenotypic
selection to make sure multiple modifier loci for opaque-2 were incorporated each
time generation advancement was carried out.
4. Carried out high-throughput DNA extraction using the BioSprint Workstation
and performed genotyping to guide field selection.
5. Principle Component Analysis (PCA) of amino acid profiles from UPLC (Ultra
Performance Liquid Chromatography) of candidate QPP lines along with
corresponding parental lines (QPM and popcorn). (in collaboration with Dr. Ruthie
Angelovici from University of Missouri Columbia, MO) DEDICATION iii
I would like to dedicate this work to all my family members who have always believed in me and have been supportive unconditionally in every aspects of my life, my advisors Dr. Xiyun Song Dr. David Holding for their generous impartion of knowledge, patient guidance during my pursuit of degrees and rigid attitude toward scientific research, and to all of my lovely friends for their companionship.
Ying Ren iv Acknowledgements
I'm indebted to Dr. David Holding for his support and encouragement. He sets good examples for me in both academia and life. I was fortunate to get onboard with this project led by him and gained great experience in performing scientific research. Great thanks to all of my committee members, Dr. Rodriguez, Dr. Clarke, Dr. Schnable and Dr. Rose who provided all the help I need to be on the right track toward graduation and a Ph.D. degree in Agronomy and Horticulture.
Many thanks to ConAgra Foods for its support in funding and providing popcorn germplasms. Also, I would like to thank the North Central Regional Plant Introduction
Station for providing QPM germplasms for research. Thanks for the collaborators who have made this research possible, Dr. Oscar Rodriguez from ConAgra, Dr.
Ruthie Angelovici and Dr. Abou Yobi from University of Missouri Columbia, Dr. Devin
Rose and Leandra Marshall from University of Nebraska-Lincoln. I feel grateful to
Christine Smith, Dr. Kyla Morton, Dr. Aixia Li and Dr. Shangang Jia for their help with field work and Mike Livingston for his help with advice on high-throughput DNA extraction and genotyping. I also would like to express my thanks to instructors whom I learned a lot from in class including Dr. Eskridge from Department of Statistics, Dr. Hyten and Dr. Amundsen from Department of Agronomy and Horticulture, Dr. Cerrutti from
School of Biological Sciences. Thanks for the knowledge and experience they’ve imparted to me.
Thanks are due to my family, my friends and my two dog companions for their v unconditional care and support in all aspects as always. They have been my tutors for love, faith and how to lead a life. My life would have been a lot less happy without any of them in it. I dedicate this dissertation to all these people in my life. vi
Funding Acknowledgement
This research was financially supported by ConAgra Foods. vii Preface
The results presented in Chapter 2, Chapter 3 and Chapter 4 were accepted for publication in Frontiers in Plant Science-Plant Breeding. (Y. Ren, A. Yobi, L. Marshall, R. Angelovici,
O. Rodriguez and D. R. Holding, “Generation and evaluation of modified opaque-2 popcorn suggests a route to Quality Protein Popcorn,” (accepted for publication in
Frontiers in Plant Science-Plant Breeding, Nov 20th, 2018).) viii Table of Contents
LISTS OF MULTIMEDIA OBJECTS
CHAPTER 1…………………………………………………………………...xi
CHAPTER 2…………………………………………………………………...xi
CHAPTER 3………………………………………………………………….xii
CHAPTER 1 Literature Review
Literature Review……………………………………………………………….1
1. Maize…………………………………………………………………………2
1.1 Maize kernel…………………………………………………………………3
1.1.1 Structure of a maize kernel…………………………………………………3
1.1.2 Kernel composition………………………………………………………3
1.2 Maize protein………………………………………………………………4
1.2.1 Zeins………………………………………………………………………4
1.2.2 Non-zeins…………………………………………………………………9
2. opaque-2 mutants and Quality Protein Maize………………………………9
2.1 opaque-2 mutants…………………………………………………………9
2.2 Quality Protein Maize (QPM) ……………………………………………20
2.3 Comparison of o2, QPM with wild type……………………………………29
3 Mechanism of amino acids change and protein quality improvement………31
3.1 Mechanism of amino acid changes in o2…………………………………31
3.1.1 Pathway for lysine synthesis and degradation……………………………31
3.1.2 Biosynthesis of other amino acids………………………………………37
3.2 Protein quality improvement………………………………………………38 ix 3.2.1 Conventional approach for protein quality improvement………………38
3.2.1.1 Marker-assisted breeding for gene introgression………………………38
3.2.1.2 QPM conversion studies…………………………………………………41
3.2.2 Application of transgenic approach for protein quality improvement………42
4. Popcorn………………………………………………………………………43
4.1 Types of popcorn……………………………………………………………44
4.2 Popcorn quality………………………………………………………………44
4.3 Popping process and mechanism for popping………………………………45
4.4 Popcorn breeding for incorporation of desirable traits……………………..51
4.5 Quality Protein Popcorn (QPP)……………………………………………55
Figures and Tables……………………………………………………………57
References……………………………………………………………………...... 67
CHAPTER 2 Design of breeding scheme for QPP development
Introduction…………………………………………………………………….108
1. Materials and ethods …………………………………………………...... 110
1.1 Plant materials……………………………………………………………110
1.2 Methods……………………………………………………………………111
2. Breeding scheme and selection procedure…………………………..…..113
2.1 Breeding cheme……………………………………………………………114
2.2 Genotypic and phenotypic selection methods applied in the breeding
process…………………………………………………………………………114
Figures and Tables……………………………………………………………117
References……………………………………………………………………..132 x CHAPTER 3 Analysis of QPP developed between three QPM and four popcorn parental lines
1. Selection of BC2F4 population with full endosperm modification………….137
2. Confirmation of o2 genotype in BC2F4 population with full modification….137
3. Amino acids profiling revealed high lysine in all six different BC2F5 o2
introgressions…………………………………………………………………..137
4. Selection of fully modified o2 popcorn can result in equivalent popping to
popcorn parents…………………………………………………………...…..140
Figures and Tables………………………………………………………...... 142
References…………………………………………………………………….156
CHAPTER 4 Discussion of the impediments of QPP breeding and solutions, its prospective and future plans
1. Discussion on the impediments of QPP breeding and solutions……………....158
2. QPP prospective and future plans………………………………….…….....160
References………………………………………………………………….165
CHAPTER 5 Appendix
1. R codes for data analysis and presentation………………………………...171
1.1 PCA analysis of protein-bound amino acids (PBAA) ………………………171
1.2 PCA analysis of free amino acids (FAA) …………………………………...172
1.3. Comparison of log2 value of ratio (o2 /wild type ratio) for 15 amino acids
using protein-bound amino acid (PBAA) measurements ……………………..173
1.4 Comparison of the relative concentration of total protein………………...175
1.5 Popping volume comparison……………………………………...………176 xi Lists of Multimedia Objects
CHAPTER 1 Introduction
Figure 1. Structure of a maize kernel (Hoseney et al., 1986).…………………………57
Figure 2. Regulatory effects of the Opaque-2 gene (Prioul et al., 2008).……………..58
Figure 3. A proposed model for PBF and O2 controlling kernel nutritional quality and
yield in maize (Zhang et al., 2016).…………………………………….…59
Figure 4. Multiple pathways in which the 35 direct regulatory targets of O2 are
involved (Mach, 2015).………………………………………………………60
Figure 5. QTLs influencing free amino acid content w mapped using the F2
population of Oh51Ao2 x Oh545o2 (Wang and Larkins, 2001).……………61
Figure 6. The aspartate family biosynthetic pathway (Galili, 1995)………………..…62
Figure 7. Enzymatic catabolism of lysine in seed (Galili, 1995).…………………...…63
Figure 8. Compatibility relationships among alleles of ga1 (Jones et al , 2015)… 64
Table 1. Thirty-five genes down-regulated in W22o2 compared with wild type with O2
binding site detected (Li et al , 2015) .. …………………………… …… 65
Table 2. Some features of maize mutants affecting zein accumulation (Motto et al.,
2003).……………………………………………………………………...…66
CHAPTER 2 Design of breeding scheme for QPP development
Figure 1. Breeding scheme for quality protein popcorn.……………………….……117
Figure 2. SDS-PAGE analysis of zein proteins (A) and non-zein proteins (B) in wild
type, o2 mutant and 12 QPMs.……………………………………………..119 xii Figure 3. Genotyping results for six randomly selected seedlings of QPM
HQPSCB.…………………………………………………………………...120
Figure 4. Zein profile of three selected QPMs (CML154Q, K0326Y, Tx807) and 11
popcorn lines (P1-P11).…………………………………………………….121
Figure 5. Screening of five pairs of o2 markers (umc1066, phi112, bnlg1200, bnlg2160,
phi057) across 11 popcorn lines and six QPMs (A), umc1066 is a codominant
marker (B) and application of umc1066 in BC1 and BC2 generations for cross
K0326Y x P3 (C).…………………………………………………………..123
Figure 6. Classification of endosperm vitreousness of F2 population (K0326Y x P2)
where Type I is fully vitreous and Type V is fully opaque.………………..124
Figure 7. Identification of completely modified poppable o2 mutants in F2 population
from cross Tx807 x P7.……………………………………………………..125
Figure 8. Confirmation of Type II and Type III opaque kernels in BC2F2 generation as
o2 mutants with low α-zein, high γ-zein compared to corresponding popcorn
parental lines in Figure 4.…………………………………………………...126
Figure 9. Presence of Type I (vitreous) o2 mutants in BC2F3 populations from partially
modified (Type II and Type III opaqueness) BC2F2 individuals.…………..127
Table 1. Evaluation of QPM parents as o2 and modifier donors.……………………129
Table 2. Evaluation of modifier transfer at F2 stage.………………………………...130
CHAPTER 3 Analysis of QPP developed between three QPMs and four popcorn parental lines
Figure 1. Kernel and zein phenotype of BC2F4 populations.…………………………142 xiii Figure 2. SDS-PAGE confirmation of o2 mutants in vitreous BC2F4 populations.….144
Figure 3. QPM, popcorn and six BC2F4 introgressions prepared for amino acid
profiling……………………………………………………………………..145
Figure 4. Confirmation of high protein quality of six QPP BC2F4 introgressions in
comparison to QPM (CML154Q, K0326Y, and Tx807) and popcorn (P3, P2,
P5 and P9) controls.………………………………………………………...146
Figure 5. PCA-biplot for QPM, popcorn and six BC2F4 introgressions using free amino
acid measurements………………………………………………………….147
Figure 6. Comparison of log2 value of ratio (o2 / wildtype ratio) for 15 amino acids
using protein-bound amino acid measurements.……………………………148
Figure 7. Comparison of relative concentration for total protein between BC2F4 QPP
introgressions and corresponding popcorn parental lines.………………….150
Figure 8. Comparison of popping characteristics between QPP introgressions with
corresponding popcorn parents.………………………………………….…151
Table 1. Amino acid profile of QPMs, popcorn parents, and six different introgressions
(BC2F4).…………………………………………………………………...... 152
Table 2. Popping volume (ml) comparison and percentages of popped kernels between
BC2F5 populations and popcorn parental lines.…………………………….154 1
CHAPTER 1
LITERATURE REVIEW 2 1 Maize
Maize is a major cereal crop ranking number one in the world grain production. There are four classes in maize, namely, popcorn, flint corn, dent corn and flour corn (primarily used for corn flour production), based on the distribution and relative proportions of hard
(glassy/vitreous) and soft (floury/opaque) starch in the endosperm. Different from the other corn types, popcorn has only a small core of soft starch near the center and the greatest proportion of hard starch (Willier and Brunson, 1927). Like other cereals, maize is nutritionally imbalanced, as the dominant seed storage proteins, the zeins, are devoid of two essential amino acids, lysine and tryptophan. Compared with the optimal amount required for human nutrition which is 5% lysine and 1.1% tryptophan, respectively, maize grain contains only 1.5-2.5% lysine and 0.25-0.5% tryptophan (Young et al., 1998). The opaque-2 (o2) mutant which has kernels with reduced zein content and increased lysine content is of great potential as a more complete source of plant protein (Mertz et al., 1964).
However, o2 mutant kernels have soft endosperm, increased susceptibility to fungal pathogens and mechanical damage and reduced grain yield. Quality Protein Maize (QPM) was developed by breeding o2 mutants with normal kernel hardness and vitreousness while retaining the high lysine content. QPM conversion studies have been carried out for many dent corn types but not for popcorn. In this study, we aimed to develop Quality Protein
Popcorn (QPP) by introgressing o2 alleles via marker-assisted selection along with QPM modifier loci via phenotypic selection while retaining an acceptable amount of popping expansion volume. 3 1.1 Maize Kernel
1.1.1 Structure of a maize kernel
A depiction of a maize kernel is shown in Figure 1 (Hoseney et al., 1986). From the picture,
the crown is collapsed because of the soft floury endosperm in dent corn. However for
popcorn, no collapse is present.
1.1.2 Kernel composition
Maize grain mainly consists of starch, oil and protein. Studies were carried out for
each of the three components. Maize seeds contain on average glucides
(compounds composed of carbon and water, Cm(H2O)n) 70.7% (of which starch
61.0%) (Martin et al., 1970). Starch consists of amylopectin (72-77%) and amylose
(21-28%). Granule bound starch synthase was studied in aspects of structure,
function and phylogenetic utility (Mason-Gamer et al., 1998). Methods for
determination of resistant starch were studied (Perera et al., 2010). Starch
biosynthesis in cereal endosperms was studied (Hannah and James, 2008). Lipids
account for about 4% of maize kernels and are stored in the embryo (Martin et al.,
1970). To study oil synthesis, genome-wide association mapping was carried out
and SNPs and candidate genes associated with oil concentration were
characterized (Li et al., 2013). It was proved that the o2 gene, which was proved
to affect protein content and lysine content, has minor effect on oil content since
its influence on oil content was weak as compared to those on lysine and protein
contents (Lou et al., 2005). Protein usually makes up ten percentage in maize
kernels (Martin et al., 1970). In terms of protein content, as endosperm and germ 4 vary in protein content and quality, considerable difference was observed between
whole grain and endosperm (Paulis et al., 1974). The germ contains practically no zein (Bressani et al., 1958; Paulis et al., 1974). Study of protein granules showed
that protein granules of maize endosperm are largest and most numerous in the
sub-aleurone cells, progressively decreasing in size from the outer to the inner cells
of the endosperm which is opposite to the grains which are progressively larger going from outer to inner cells (Duvick, 1961). The development of these protein granules was observed between 15-20 days after pollination (DAP) (Duvick,
1961). Later, the average amount of total protein and of 17 out of 20 amino acids w determined in normal ma ze (Rysavá, 1994). To increase quantity and quality of protein in maize, studies were carried out for improvement of total protein and lysine respectively.
1.2 Maize protein
Proteins of maize kernel can be differentiated based on their solubility (Osborne,
1897). The proteins are categorized into albumin, globulin, prolamin, and glutelin.
Since the discovery and designation of zein fraction in 1821, Weyl found a
different fraction of protein can be extracted in 10% sodium chloride solution
(Weyl, 1877) and can further be classified into albumins and globulins based on
the solubility in water and/or salt solution. Glutelin, another category of protein,
can be extracted by alkaline solution (Osborne et al., 1914).
1.2.1 Zeins
Prolamins, known as zein proteins in maize (kafirin in sorghum), are storage 5 proteins common to all cereal grains and contain large amounts of the amino acids
including glutamine, proline, leucine, and alanine and are of relatively poor
nutritional quality (Wall and Paulis, 1978). In 1821, protein soluble in 70% alcohol
was extracted by Gorham from corn meal and was termed as zein (Gorham, J.,
1821). Although zein has been detected in vacuole aleurone and embryo (Reyes et
al., 2011; Tsai, 1979), the majority of zein are stored in endosperm and zeins account for 40-50% of endosperm protein of maize kernels. Zein can be used in fiber, adhesive, coating for food and pharmaceutical use, ceramic, ink, cosmetic, textile, chewing gum and biodegradable plastics (Anderson and Lamsal, 2011;
Shukla and Cheryan, 2001). The prolamins are synthesized on the rough endoplasmic reticulum (rER) during seed development (Zhang et al., 2013). After synthesis, nascent zeins are translocated into the lumen of the rER via the targeting effect of the signal peptides, which are short peptide at the N-terminus of the newly synthesized proteins. In the rER, signal peptides are cleaved and the processed zeins are sequestered by different cellular pathways for protein body formation
(Galili et al., 1993).
The zeins have been classified based on the relatedness of their primary gene and
amino acid sequences. Zeins comprise of four types α-(19kD and 22kD), β-15kD;
16kD and 27kD γ-zein; 10kD and 18 kD δ-zeins which can be separated, after selective solubilization in 70% ethanol and then evaporation, by polyacrylamide gel electrophoresis (Wall and Paulis, 1978; Coleman and Larkins, 1999; Esen,
1987; Leite et al., 1999). There is a multi-gene family for α-zein proteins (Lending and Larkins, 1989). α-zeins which make up>70% of zein proteins are encoded by 6 genes clustered across several chromosomes (Pirona et al., 2005). Zein genes were
located on different chromosomes (chromosome 1, 4, 7) in the maize genome
(Gibbon and Larkins, 2005). Studies of the amino acid sequence of zein protein
revealed that zeins are actually devoid of lysine and tryptophan and are also low
in arginine and aspartic acid (Geraghty et al., 1981; Baudet et al., 1966; Mossé et
al., 1966; Wall and Paulis, 1978). Methylation state of the storage protein genes of
maize was investigated in both somatic tissues and developing endosperms
(Bianchi and Viotti, 1988). Expression of zein genes are expressed between 10 to
12 days after pollination (Zhang et al., 2015). Although these genes were proved
to be transcribed only in endosperm cells, it was found that prolamins can be
delivered into the vacuole of maize aleurone cells (Reyes et al., 2011).
Hypomethylation of zein sequences were shown after southern blotting of DNA
restricted by methylation-sensitive enzymes (Bianchi and Viotti, 1988).
α-zein
α-zeins are by far the most abundant fraction and comprise up to 80% of the total zeins.
Among the four subfamilies of zein (α-, β-, γ-, δ-), all but α-zein are encoded by single or low copy gene loci (Pirona et al., 2005). The 22-kD zein family in maize has 23 members. Distance analysis of these members was carried out for an inbred maize,
BSSS53 and showed that the ancestral z1C gene arose before 11.5 million years ago where the allotetraploidization of maize was estimated to take place (Song et al., 2001).
β-zein
β-zein is another subfamily of zein. or maize, segregates for - - 7 kD molecular weight.
γ-zein
The 27kD γ-zein gene expression was proved to occur as early as 10 DAP (Zhang et
al, 2015). RNAi suppression has shown that 27kD γ-zein controls protein body initiation while other γ-zein family function in protein body expansion (Guo et al.,
2013). Quantitative extraction of zein proteins revealed that QPM germplasms have
high γ-zein (Wallace et al., 1990). γ-zein was reported to play a haploinsufficient role
in opaque-2 endosperm modification (Yuan et al., 2014).
δ-zein
The location where δ-zein exists in the protein bodies were revealed by
immunocytochemical localization (Esen and Stetler., 1992).
Formation of protein bodies
Zein proteins accumulate and result in the formation of protein bodies which was
confirmed by microscopic studies (Christianson et al., 1969; Duvick, 1961). The
protein bodies were shown to develop from cisternae of the rER (Khoo and Wolf,
1970). From the observation of the polyribosomes seen on the surface of protein bodies in maize, Burr et al proposed that zein might be synthesized and deposited on the protein bodies (Burr and Burr, 1976). It was shown by electron microscopy
that the protein bodies were surrounded by membranes which are continuous with rough endoplasmic membranes indicating protein bodies are within the rER
(Larkins and Hurkman, 1978). Lending et al observed the size of protein bodies in 8 developing endosperm and illustrated that α-zein resides in the central region of the protein bodies whereas β-and γ-zein are peripheral (Lending and Larkins,
1989). The spatial distribution of zein proteins within the starch granule was
determined (Mu-Forster and Wasserman, 1998). The development and importance of zein protein bodies in maize endosperm was summarized (Holding and Larkins,
2006). Starch granules are encapsulated by prolamin proteins. A method, mTZM
(modifed turbidimetric zein method) was able to quantify starch granule encapsulation by hydropgobic prolamin proteins in corn (Larson and Hoffman,
2008). Substantial loss of 22-kD α-zeins by z1C RNAi was observed which resulted in protein body budding structures, indicating the necessity of 22-kD zein in normal formation of protein body (Wu and Messing, 2010).
Zeins are of low protein quality
Zeins are rich in glutamine (21%-26%), leucine (20%), proline (10%) and alanine
(10%) whereas they are devoid of lysine and tryptophan (Tripathy et al., 2017).
Because of the absence of tryptophan and lysine, zeins are not ideal for human
consumption (Shukla and Cheryan, 2001). Via in vitro mutagenesis, cDNA of the
19kDa α-zein gene were modified such that several original amino acids were substituted for lysine. Capped, polyadenylated mRNAs were collected after transcription in Xenopus oocytes and injected to stage 6 oocytes which result in successful synthesis and processing of zein proteins and formation of structures resembling maize protein bodies, suggesting that amino acid substitution for lysine had no effect on the ability of α-zein protein to be incorporated into protein body 9 in the frog Xenopus oocytes system (Lending et al., 1992). Similarly, in sorghum
kafirin is the storage protein. It was shown that a single-point mutation in the signal
peptide of kafirin conferred reduction of lysine poor kafirins resulting in sorghum
of high value (Wu et al., 2013). CRISPR/Cas9-mediated editing of an alpha-kafirin
gene family resulted in increased digestibility and protein quality in sorghum (Li et al.,
2018).
1.2.2 Non-zeins
Non-zein proteins were compared between nearly isogenic opaque endosperm mutants
and corresponding wildtypes. In total, 2762 proteins were identified across all
endosperm samples with replications. By carrying out in silico dissection of the most
significantly changed proteins in o2 and fl2 compared with wildtype, it was suggested
that protein compositional changes may contribute to the difference in lysine content
(Morton et al., 2015).
2. opaque-2 mutants (o2) and Quality Protein Maize
2.1 opaque-2 mutants
opaque-2 mutants were characterized with an opaque appearance by which they were differentiated from normal kernels. The name of opaque mutants is because of the light transmission property of the mutant kernels. Studies were carried out for composition difference of opaque 2 mutants with normal maize. Comparison of starch structure across endosperms of wild type (B46wt, m14wt), opaque-2
(B46 o2, M14 o2), and a QPM inbred line led to the conclusion that o2 maize
(including QPM) contained less starch (66.9%) than those of B46wt (73.0%) and 10 o2 maize would be hydrolyzed faster than normal counterpart (Hasjim et al., 2009).
Comparison between normal corn and o2 mutant using electron microscopy showed
the different protein network inside endosperm cells after starch digestion with α- amylase (Wolf et al., 1967). While normal corn has an amorphous matrix with granules rich in zein protein, o2 corn has smaller granules with size being 1/20 of that of the normal corn constituting lower zein protein. The opaque-2 (o2) mutant which has kernels
characterized with reduced zein content and increased lysine content offered potential as a
more complete source of plant protein (Mertz et al., 1964).The lysine increase in opaque-
2 mutants is a result of low zein content with higher content of other proteins which are rich in lysine (Mossé et al., 1966). Since this discovery, characterization of the mechanism conferring the increased protein quality of this mutant has begun for maize
protein quality improvement.
Difference in lysine and tryptophan content between wild type and o2
genotypes
Lysine measurement of both mutant and normal maize showed that lysine content
in o2 mutants (3.3-4.0%) was more than twice that of normal maize (1.3%). Amino acids composition was determined for kernels of both phenotypes (normal and
opaque-2) from the backcross ear (O2o2 x o2o2) and a 69% increase was found in o2 endosperm compared with the normal counterpart. W22 floury endosperm mutant was analyzed as containing 3.4 grams of lysine per 100 grams of protein compared with the wildtype W22 which contains 2.1 grams of lysine per 100 grams of protein with opaque 2 and floury 2 mutant as a control giving lysine 11 content as 3.65 and 3.2 grams per 100 grams protein, respectively (McWhirter,
1971). Lysine content was compared between entirely opaque kernels and partial opaque (½ opaque : ½ translucent) which showed no significant difference (Paez et al., 1969). For the partial opaque kernel (½ opaque : ½ translucent), no difference was found between translucent and opaque fractions of the endosperm.
Determination of lysine and tryptophan
A 3:1 ratio of lysine to tryptophan was reported (Hernández and Bates, 1969; Bjarnson
and Vasal, 1992; Vivek et al., 2008). With this ratio between lysine and tryptophan, it
is reasonable to measure only either one of the two in QPM breeding which is necessary
as they cannot be co-extracted by with acid or alkaline hydrolysis methods. Opienska-
Blauth developed a tryptophan measurement method for use in biological materials
(Opienska-Blauth et al., 1963). Based on the simplicity of this method, a modified
version was developed (Hernández and Bates, 1969). In a survey of 364 white endosperm maize accessions consisting of 195 dent, 28 floury, nine popcorn and 132 flint types, 12 accessions had lysine values larger than 0.22% (Zuber et al., 1975).
Among the nine white endosperm popcorn accessions, none exceeded 0.22% in endosperm lysine. Determination of tryptophan and lysine in endosperms was carried out in normal maize and o2 maize and it was found that on average normal maize has about 0.45g of tryptophan and 1.8 g lysine per 100g of protein, o2 maize has about
0.85 g of tryptophan and 3.6g of lysine per 100g of protein) (Villegas and Mertz,
1971). A reliable and inexpensive method for determining protein-bound tryptophan
in maize kernels based on colorimetric method was developed (Nurit et al., 2009). To 12 evaluate lysine on a large number of whole kernel and endosperm meals, a rapid
turbidimetric assay for zein was developed (Paulis et al., 1974). Also a highly
significant negative correlation (-0.76 for endosperm across 12 samples and -0.93 for
whole kernel across 23 samples) was found between lysine content of the protein and
zein content of the meal. To determine tryptophan, microbiological assays, chromatographic techniques, spectrophotometric analysis and chemical estimations following alkaline or enzymatic hydrolysis were carried out. A correlation (r=0.05) was confirmed with the regression equation between per cent of tryptophan and lysine in protein. With the advent of liquid chromatography tandem mass spectrometry (LC-MS/MS), compostition of amino acids can be determined. Amino acid profiling was carried out for MUDISHI3, S1 and S2 inbred lines via the facility at the University of Missouri Agricultural Experiment Station
Chemical Laboratories (ESCL) (Nkongolo et al., 2015). Angelovici et al carried out analysis for amino acids of Arabidopsis seeds using LC-MS/MS system
(Angelovici et al, 2013). UPLC-MS/MS was adopted for amino acid profiling for sorghum line Tx430 and CRISPR/Cas9-edited T2 transgenic lines (Li et al., 2018).
Opaque 2 gene
Opaqueness of the o2 mutant is a recessive trait controlled by opaque 2. Isolation
of the Opaque-2 gene was achieved via transposon tagging method (Schmidt et al.,
1987). The O2-specific cDNA clones were screened out from cDNA library prepared
from a wild-type endosperm (A69Y) and O2 sequence was determined (Hartings et al., 1989). The Opaque2 gene (O2) encodes an endosperm-specific transcriptional 13 activator which is specifically expressed in the subaleurone layers of maize endosperm
during seed development. (Schmidt et al., 1992). Molecular evolution of the O2 gene
was studied by comparing O2 sequence including 5’ UTR and 3’non-coding regions
across a set of cultivated and teosinte accessions (Henry et al., 2005). 106 polymorphic
sites were found from the region consisting of 1976 nucleotide positions. The
molecular diversity was confirmed to be high for o2 compared with other transcription factors in maize (Henry et al., 2005). Diverged copies of the O2 gene were reported as a result of segmental duplication in the progenitor genome of maize (teosinte)
(Xu and Messing, 2008). Study of 21 maize inbred lines at the O2 locus revealed
high level of polymorphism: 109 sites variable in non-coding regions and 159
in protein-coding regions (Henry and Damerval, 1997).
O2 is a transcription factor
The action of O2 has been described at the molecular level. The O2 protein is localized
in the cell nucleus. The O2 gene encodes a DNA binding protein with a leucine
binding motif. This protein can bind the 5’ flanking sequences of 22-kD α-zein
genes regulating their expression. As this binding is thought to be part of the
normal transcription process involving the Opaque2 encoded protein and other
transcription factors, a mutant would be expected to reduce but not completely
eliminate the transcription of multiple α-zein genes. After its sequence was
determined, Opaque2 was confirmed to encode a protein with structural homologies to transcriptional activators in that it contains a domain similar to the leucine zipper motif as previously found in transcriptional activators such as GCN4 14 and C/EBP (Hartings et al., 1989). The o2 mutation of maize differentially reduces
transcription of subfamilies of zein genes (Kodrzycki et al, 1989). The
transcription of genes encoding α-zeins is inhibited significantly especially the 22- kDa α-zeins whose expression is almost completely abolished whereas other zein
gene subfamilies were affected to a lesser extent. O2 encodes a protein with leucine
zipper motif that can bind to zein DNA (Schmidt et al., 1990). It was observed that
a substitution of a lysine residue for an arginine residue within the bZIP domain of
O2 gene has abolished specific DNA binding between the mutant opaque2 protein to the zein promoter fragments (Aukerman et al., 1991). Vice versa, mutations in the zein (22-kD and 27-kD) promoters were confirmed to affect transactivation by the O2 protein (Ueda et al., 1992). Fourteen proteins affected by mutant allele (o2) in maize were identified across two different backgrounds (wild type v.s. a dent type W64Ao2 and wild type vs a flint type F2o2) via 2-D PAGE (Damerval and
De Vienne, 1993). The protein was shown to activate the transcription of the 22- kDa α-zein (Schmidt et al., 1992), and 14 kD β-zein genes (Neto et al., 1995), together with the β-32, a ribosome inactivating protein (RIP) (Lohmer et al., 1991)
and cyPPDK1 genes (Maddaloni et al., 1996). The PBF (prolamin-box binding
factor) was reported to bind the prolamin box in zein gene promoters and interact
with O2 (Vicente-Carbajosa et al., 1997). A set of proteins (36 polypeptides, eight of which consist of six zein proteins, b-32 and a high molecular globulin) were identified as being consistently affected in o2 mutants in all seven backgrounds which are (W64A, F2, F7, W22, W23, B37 and OH43) (Damerval and Le Guilloux, 1998).
In order to dissect the regulatory network of O2, transcriptome and proteome analysis 15 were carried out to show the relationships between polymorphisms of O2 locus and
the expression of genes related to its regulatory network (Lefèvre et al., 2002). Opaque endosperm mutantions (o1, o2, o5, o9, o11, Mc, DeB30 and fl2 in W64A background) were reported to create changes in patterns of gene expression (Hunter et al., 2002).
In the mutants, 60 most up-regulated (more than 3-fold) genes are related to stress
responses, molecular chaperones and protein turnover but also to amino acid
synthesis. Of the 66 down-regulated genes, many are involved in carbon,
carbohydrate metabolism and branched chain amino acids (Hunter et al., 2002).
Maize proteins whose accumulation was affected by the o2 mutation in maize
kernels were summarized (Motto et al., 2003). In a study observing o2 mutant alleles (o2T, o2-52, o2It, and o2-676), only wild-type O2 and mutant-defective o2 polypeptides bearing the Leu-zipper are able to form complexes in order to bind target sizes (Gavazzi et al., 2007). A comparison of transcript profiles between eight pairs of o2 and wild type counterpart was made in different genetic backgrounds and two scenarios which are differentiated expression in specific genotypes and different degree of differential expression across all backgrounds were generalized (Jia et al., 2007). Direct effect of o2 on PPDK gene expression was studied (Prioul et al., 2008). The relationship between O2 and PPDK is epistatic. Regulatory effects of the Opaque 2 gene was summarized which is shown in Figure 2 (Prioul et al., 2008). Identification of seed-specific transcription factor was carried out in Arabidopsis (Le et al., 2010). Opaque2 is important in the regulatory network in maize kernel development (Fu et al., 2013). bZIP proteins are characterized as having a conserved bZIP domain, which is 60-80 amino acids in
16 length with two basic regions for DNA binding and leucine zipper dimerization
region (Zhang et al., 2014). Roles of bZIP genes in rice were described (Zhang et
al., 2014). O2 was among the master regulators (besides O2 heterodimerizing
proteins (OHP) and prolamine-box binding factor (PBF)) of zein storage protein synthesis (Zhang et al., 2015). opaque 2 was also confirmed to be involved in starch synthesis along with another transcription factor PBF via regulation of critical components in starch biosynthetic enzyme complex (Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase (SSIII)) with the proposed model shown in Figure 3 (Zhang et al., 2016). The o2 mutation causes a
suppression of specific members of the 22-kD zein class, due to a lower rate of
transcription. The leucine zipper domain of opaque 2 protein binds to the promoter of 22-kd zein gene. The mutant protein does not show specific recognition of zein promoter fragment. This indicates that its function is direct instead of through other regulatory factors. bZip proteins can interact with a G-box DNA element
(CACGTG, G-box). G-box sequences reside in the promoters of many genes. The analysis of the G-box as a regulatory element has been complemented by the isolation of a family of bZip proteins that specifically interact with this element.
The regulatory network of O2 was studied by RNA sequencing tandemed with chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-
Seq) (Li et al., 2015). RNA seq analysis alone revealed 1605 differentially expressed genes (DEGs) whereas ChIP-Seq analysis dected 1686 binding sites of
O2 across about 1143 genes. In total, thirty-five O2-modulated target genes were shared between the two aforementioned analyses which is shown in Table 1 (Li et 17 al., 2015). O2 transcriptional regulation in maize endosperm was depicted in
Figure 4 (Mach, 2015). Since the O2 gene regulates many genes, the o2 mutation
results in the many phenotypic characteristics (pleiotropic effect of O2) such as
lower grain yield (10%-15% less), slower height increase and increased
succeptibility to ear-rot-inciting fungi (Lambert et al., 1969; Nass and Crane, 1970;
Loesch et al., 1976).
Zein expression controlled by pbf1 (prolamin box binding factor),
ZmMADS47, OHP1 and OHP2
Expression of zein genes are also controlled by a transcription factor 1 (pbf1) (Lang
et al., 2014). From RNAi lines of ZmMADS47, down-regulation of α-zein and 50kD
γ-zein were observed indicating that zein gene transcription can also be regulated
by ZmMADS47 (Qiao et al., 2016). O2 heterodimerizing protein 1 (OHP 1 on
chromosome 1L) was reported to be able to bind to O2 target site in 22-kD zein
genes as homodimers and also bind with O2 as heterodimeric complex (Pysh et al.,
1993). Another O2 heterodimerizing protein, designated as OHP 2, on
chromosome 5S was reported to be 88% identical to OHP1 (99% identical within
the bZIP motif) and is presumed to have similar properties to OHP1.
O2 in-gene markers
Three in-gene markers have been reported, which are umc1066, phi057 and phi112.
Among these, phi057 and umc1066 are of co-dominant type while phi112 is dominant. umc1066 is located in Exon 1. phi057 is located in Exon 6. phi112 is located
in Opaque 2 leader sequence between the G-box and 3’ upstream ORF. phi057 was
18 reported to show polymorphism of two bands being 169 bp (QPM allele) and 159 bp
(normal maize) (Magulama and Sales, 2009). This is consistent with the result from
another study where phi057 amplified 170 bp fragment in CML144 and 160 bp in
ZPL5 (Kostadinovic et al., 2014). For umc1066, amplicon size for CML144 was 150bp
whereas it was 160-170bp for ZPL5 (Kostadinovic et al., 2014). phi112 exhibited
dominant polymorphism between the two parents; no band present in CML144 and
170bp DNA fragment in ZPL5 (Kostadinovic et al., 2014).
The polymorphism of sequence at loci phi057, phi112 and umc1066 varied across
different germplasms. Because of this variation, confirmation is necessary before their application. In QPP breeding, we screened all five o2-related markers for verification
of dominance or codominance type and amplicon size in our germplasms. Beside
simple sequence repeats (SSRs), restriction fragment length polymorphism (RFLP)
showing polymorphisms between the wild type allele O2 and the mutant allele o2 were
reported to identify o2 genotypes in segregating populations from the cross QPM by
Tx5855 (Kata et al., 1994).
Application of O2-related markers
O2-related markers were applied in foreground selection in breeding projects. 14
inbred o2 lines and a wildtype line were used to investigate the allelic variations at
the three marker sites (umc1066, phi057, phi112) within the o2 gene (Yang et al.,
2004). phi057 and phi112 were used to successfully detect QPM germplasms
among S0 plants (Jompuk et al., 2006). These three markers were used for
amplification of the o2 locus as an indicator of improved quality protein maize
19 (Orlovskaya et al., 2016).
Other mutants beside o2
Beside opaque2 mutants, other mutants were characterized some of which can alter the amino acid profile of maize endosperm protein or have similar opaque endosperm.
A new source of maize opaque 2 mutant was characterized (Peöev et al., 1973).
Defective endosperm De-B30 is a dominant maize mutation that depresses zein synthesis in the developing endosperm. This mutation maps to a cluster of 19-kD
α-zein genes. It was proved that a defective signal peptide (proline replaces serine at the 15th position of the signal peptide) in the 19-kD α-zein protein which can cause unfolded protein response and result in opaque endosperm phenotype (Kim et al., 2004). For the dominant Mucronate (Mc) mutant, Mc gene was reported to
encode a 16-kD γ-zein with a frameshift mutation (Gibbon and Larkins, 2005).
opaque 1 mutant of maize has an opaque endosperm while maintaining a normal zein
protein content. The normal Opaque 1 was isolated and confirmed to encode a myosin
XI motor protein which is required for endoplasmic reticulum motility and protein
body formation in maize endosperm (Wang et al., 2012). dek mutations occurred in
kernels that fail to initiate or complete grain-filling (Neuffer and Sheridan, 1980;
Scanlon et al., 1994). floury1 gene was confirmed to encode a novel endosplamsic reticulum protein involved in zein protein body formation (Holding et al., 2007).
floury 2 was confirmed to have altered amino acid pattern with increased lysine
and methionine (Nelson et al., 1965). floury2 was found to be caused by a defective signal peptide in a 22-kD α-zein protein (Coleman et al., 1997). opaque 6 and floury 20 3 were later identified and characterized with high lysine resulting from the
decrease in the ratio of zein to other protein fractions (Ma and Nelson., 1975).
Floury4, a semi-dominant opaque mutant was identified and proved to disrupt
protein body assembly (Wang et al., 2014). opaque 7 mutant was confirmed to be
defective in an Acyl-CoA Synthetase-like protein (Miclaus et al., 2011). opaque
15 mutation, mapped near the telomere of Chromosome 7L, was identified with a
2- to 3- fold reduction in γ-zein mRNA and protein synthesis of the 27-kD γ-zein
(Dannenhoffer et al., 1995).
The inheritance and effect on zein accumulation of these mutants were summarized
in Table 2 (Motto et al., 2003). Effects of these maize mutants on zein accumulation were summarized (Pirona et al., 2005). About inheritance pattern of these traits, the opaque mutants are recessive (o1, o2, o5, o9-11, o13, 016, o17),
the floury mutations are semi-dominant (fl-1, fl-2 and fl-3) whereas ‘Mucronate’ and
‘Defective endosperm’ are dominant mutations (Tripathy et al., 2017).
2.2 Quality Protein Maize (QPM)
QPM was developed to alleviate protein malnutrition (Nuss and Tanumihardjo,
2011). A positive relationship between high γ-zein levels and kernel hardness /
vitreosity in QPM kernels was reported (Wallace et al., 1990). Selection of
opaqueness or vitreousness can be considered as selection of kernel hardness/semi-
hardness. It was found that there is more total zein in hard endosperm (Paiva et al.,
1991). For measurement of kernel density, it was proved that kernel density can be
predicted via single kernel near infrared spectroscopy (Armstrong and Tallada., 21 2012). The model for prediction of kernel density is of reasonable accuracy with th e
R square being 0.79. Maize kernels consist of variable proportions of hard and soft
endosperm. Flint maize is a type of hard endosperm whereas floury type maize has
a soft endosperm and dent corn has variable proportions of hard and soft
endosperm (Pratt et al., 1995). The effect of protein composition on endosperm hardness is genotype-dependent (Pratt et al., 1995). It was reported that altered
starch structure (shorter amylopectin branches and increased starch-granule
swelling) is associated with endosperm modification (Gibbon et al., 2003). 27kD γ- zein content was found to be related to endosperm modification in quality protein maize (Lopes and Larkins., 1991). γ-zeins (27-kD and 16-kD) are proved to be essential for endosperm modification in quality protein maize (Wu et al., 2010).
Modifier evaluation and Chi-square test
The term modifier genes has been used to describe the genes which convert the soft endosperm of o2 genotype to a hard (vitreous / glossy, corneous) type of
endosperm. VI (Vitreous Index) was proposed to determine the vitreousness of
maize kernels (Louis-Alexandre et al., 1991). This methodology involves kernel
sectioning (longitudinal section through the germ, longitudinal section parallel to the
germ face through the largest dimension of the kernel, transverse section through the
median of the longest dimension of the kernel) and measurement of the vitreous and
the total endosperm areas. Good correlation (with r ranging from 0.75 to 0.82) was
observed between true vitreousness (estimated as the percentage of vitreous relative to
total endosperm dry weights) and the calculated VI (Louis-Alexandre et al., 1991). A 22 good strategy to evaluate modifier effect is to put all the modifier to the o2
background, without the need to take segregation into account in the segregating
populations with different percentages of genotypes (O2O2, O2o2 and o2o2). In our study, we aimed to evaluate modifier transfer from QPM germplasms to popcorn
germplasm by evaluation of endosperm vitreousness / opaqueness. By the degree of
kernel opaqueness on light box, we classify kernels into five groups with Type I being
fully vitreous, Type II being approximately 25% opaque, Type III being
approximately 50% opaque, Type IV being approximately 75% opaque and Type
V being fully opaque according to the reported classification (Vivek et al., 2008).
If o2 endosperm is well modified, level of kernel opaqueness should be from Type I to Type II opaque. Specifically, for ears not fully filled with kernels, evaluation of
kernels with opaqueness can still be carried out. By evaluating the opaqueness gradient
of all kernels showing ≥5% opaqueness and estimating the ratio for each opaqueness
classification, it is possible to evaluate modification. The accuracy of the selection
according to endosperm phenotype may be affected by hetero-fertilization, which
is considered to result when the polar nuclei and the egg fuse with male nuclei of
differing genetic constitution (Gao et al., 2011). These may be up to a 5%
difference between the selections based on endosperm phenotype and plant
genotype. Specifically, in a population segregating for O2O2, O2o2, o2o2
genotypes, chi-square test can be used to evaluate the effect of modifiers which is
indicated as the deviation of phenotypic ratio (opaque v.s. vitreous kernels ) from
the genotypic ratio (‘o2o2’ v.s. ‘O2O2 and O2o2’) assuming no modifier effect. A segregation ratio of 3:1 was observed as expected between kernels of vitreous and 23 opaque phenotype in the F2 population from normal and typical (unmodified) o2
(Bjarnason et al., 1976). Exceptions with more translucent kernels than expected
suggested the presence of modified o2 mutants that were indistinguishable from normal kernels. By selection, modifiers of o2 which give hard kernel types have been
developed. These modifiers have been shown to specifically influence certain classes
of storage proteins in the kernel. Chi-square segregation ratio testing was applied to evaluate effects of modifiers for endosperm modification by testing the significance of departures from expected ratios. Chi-square values were calculated
to indicate whether segregation ratio was significantly deviated or not among doubled
haploid (DH)populations and F2 populations (Sayed et al., 2002). A Chi-Square test
can be used to see whether the deviation from expected ratios is significant or not
(Collard et al., 2005).
Study of o2 modifiers
Modification is conferred by many genes (Bjarnason et al., 1976; Vasal, 1975). A set
of modifier genes in the o2 genetic background are known to improve hard and vitreous
kernels (Bjarnason et al., 1976; Burnett and Larkins, 1999; Ortega and Bates, 1983).
Although the instability of genetic modifiers under different environmental conditions was observed, it was suggested that researchers should not be concerned
about the stability of modifiers if enough cycles of selection were carried out
already and variation for endosperm modification has been reduced to a minimum
(Vasal et al., 1980). Two o2 modifiers were identified on Chromosome 7, one mapped
near telomere of chromosome 7L and the other one mapped near the 27 kD γ-zein locus 24 (Lopes et al., 1995). Mapping of Quantitative Trait Loci (QTLs) for endosperm modification was carried out and two major QTLs were dentified near centromere and telomere respectively on the long arms of Chromosome 7 (Lopes et al., 1995). The effect of endosperm modifiers on normal genotypes is shown as increased content of
γ-zein and no increase in lysine content (Moro et al., 1995). Activity of o2 modifiers was confirmed to be affected by the genetic background (Lopes and Larkins., 1995).
QTLs for both endosperm modification and amino acids was carried out and were mapped to Chromosomes 3, 5, 6, 8 and chromosomes 7 and 8, respectively
(Gutiérrez-Rojas et al., 2010). The starch from QPM is different from its wild type
and o2 counterparts (Gibbon and Larkins, 2005). Association of altered starch structure with endosperm modification was observed (Gibbon et al., 2003). The comparison between o2 and modified o2 was carried out in terms of starch structure and organization (Gibbon and Larkins, 2005). Modification might possibly be because of the open starch granule structure which enables more γ-zein to penetrate the granule matrix resulting in a more extensive cross-linked proteinaceous network than the unmodified counterpart (Gibbon and Larkins, 2005). The pullulanase gene, a starch metabolic gene, was reported to be associated with increased digestibility when in the low frequency allele type (Gilding et al., 2013).
Strong positive correlation was found between pullulanase (R2= 0.3762, p<0.05)
with kernel vitreous and similarly starch synthase III was also an important factor
for kernel vitreousness in quality protein maize (Wu et al., 2015).
27-kD γ-zein locus 25 Homologous recombination was proven to take place and create new allelic form
at the 27-kD locus (Das et al., 1991). A transcriptional activator binding site was
identified in the 27-kDa zein promoter region (Ueda et al., 1994). Sequence of the
27-kD γ-zein was proved to be variable (Das et al., 1991). Duplicated allele was
found from maize inbred line A188 and W22, whereas single copy allele was found
in inbred line W64A. γ-zeins are necessary for modification (Yuan et al., 2014;
Wu et al., 2010). Multiple lines of evidence suggest that an increase in gene expression and protein accumulation of 27-kD γ-zein plays a major role in modification in QPM and this gene resides within the major QTL on Chromosome 7 (Geetha et al., 1991;
Holding, 2014; Holding and Larkins, 2008; Wu et al., 2010). γ-zein was reported to
play a haploinsufficient role in opaque-2 endosperm modification (Yuan et al., 2014).
Recently, it was verified that the 27-kD γ-zein protein increase is conferred by a
duplication event at 27-kD γ-zein locus (Liu et al., 2016).
Modifiers for amino acids
An increased level of free amino acids was reported in maize mature endosperm across
several o2 inbreds compared with wild type (Wang and Larkins., 2001). QTL mapping
analysis of the F2:3 family from cross between Oh545o2 and Oh51Ao2 identified four
loci on chromosomes 3, 4, and 7 (Figure 5) accounting for 46% of the phenotypic
variation in free amino acid (FAA), which are coincident with the genes encoding the
mono functional Asp kinase 2, the bifunctional Asp kinase homo-Ser-dehydrogenase-
2, and a cytosolic triose phosphate isomerase 4 (Wang and Larkins, 2001). Amino acid modifiers were reported for improvement of lysine and tryptophan (Krivanek 26 et al, 2007). Markers for amino acid modifiers (bnlg2248, phi072, bnlg 1633, bnlg1382, phi075 and mmc0241) and markers for endosperm hardness modifier gene
(umc1216) were used to create allelic pattern between normal maize and o2 maize
(including QPM) (Ignjatovic-Micic et al., 2009).
QPM diversity and QPM improvement
Comparison of QPM and common hybrid maize in chemical composition showed
similarity in terms of total carbohydrate, protein, crude fat, fiber, phosphorus,
potassium, calcium and magnesium except for two limiting amino acids lysine and
tryptophan (Paes and Bicudo, 1994). Similarly, the relationship and similarities of
94 maize elite inbreds were determined from genotyping using SSR markers
(Senior et al., 1998). DNA microsatellites have been applied to infer genetic
structure and diversity among 260 maize inbred lines (Liu et al., 2003). 30 unique
SSR alleles were detected to differentiate between QPM inbreds (Bantte and
Prasanna, 2003). In the study of SSR polymorphism (using 43 SSR markers) among a set of 23 QPM lines, QPM can be effectively differentiated (Bantte and Prasanna,
2003). Assessment of genetic diversity was carried out in 63 QPM lines using 48 SSR markers and grouping was performed according to the similarity matrix (Krishna et al., 2012). Genetic diversity between 33 inbred lines and seven quality protein maize lines were evaluated by 14 primers (Hemavathy, 2015). For QPM improvement, development of β-carotene-rich QPM hybrids were achieved through marker-assisted introgression of crtRB1 gene conferring high β-carotene (Gupta et al., 2015). 27 Concerns for QPM application
Application of QPM requires rigid prevention from cross-pollination with wild type
maize. QPM has a recessive o2 genotype and its grain quality will be reduced to wild
type levels when pollinated by normal endosperm maize (Machida et al., 2012). Cross-
pollination between o2 maize and wild type maize will result in poor protein quality
and should be avoided. Significant cross-pollination (63% to 83%) was reported when
QPM was planted within ten meters of yellow normal endosperm maize (Machida et al., 2012) whereas no significant cross-pollination was observed when QPM was planted at 12 meters from normal endosperm maize (Twumasi-Afriyie et al., 1996).
There are also several concerns for maintaining the protein quality of QPMs.
Several S1 and S2 inbred lines from a released QPM variety (MUDISHI 3) showed significant decrease of lysine and tryptophan compared with parental variety, which suggests the necessity of development of composite lines (Nkongolo and
Mbuya, 2015). It was pointed out that one limitation in the application of QPM
maize is the unavailability of suitable QPM hybrids for farmers (Agrawal and
Gupta, 2010). The most reliable way to maintain high lysine level is to buy new
hybrid seeds for each planting season (Lauderdale, 2000). To further increase lysine and tryptophan content, the combination of DNA marker and transgenic approaches are expected (Agrawal and Gupta, 2010). For development of QPM
hybrids, QPM conversion of both parental lines is essential. Surender et al carried
out QPM conversion for BML6 and BML7, which are the parental lines for hybrid
DHM 117 (Surender, 2017). Similarly, parental lines of BH660 were converted to 28 QPM version BH660Q (Worku et al., 2012). Because the protein quality trait is a recessive trait conferred by o2 in the homozygous state, contamination from normal maize germplasm should be strictly avoided and special techniques for seed productions should be implemented for QPM production in terms of seed purity
(Krivanek et al., 2007). Another proposed solution is to blanket an area with QPM
to avoid cross pollination between maize (Lauderdale, 2000). It was noted that
some QPM genotypes show less modified grains when under drought stress
(Ngaboyisonga et al., 2009) and attention should be paid to this in future breeding programs.
Biochemical and agronomic performance of QPM hybrids in temperate regions was
studied in terms of kernel modification, tryptophan and total protein contents, quality
index (QI) (defined as percentage of the ratio of tryptophan to protein) and grain yield
(Ignjatovic-Micic et al., 2013). To ascertain the introgression of modifiers for endosperm modification and amino acid, it is expected that markers be developed which are closely linked to these loci (Krivanek et al., 2007). Accuracy of methods
for grain amino acid content is a critical component of QPM breeding (Krivanek
et al., 2007). Methods for protein quality evaluation used in the past include colorimetric method for tryptophan (Hernández and Bates., 1969), enzyme linked
immunosorbent assay (ELISA) for protein synthesis factor EF-1α (Moro et al.,
1996) and protein quality assessment via ninhydrin reaction where various
chromophores can be formed and analyzed (Zarkadas et al., 2000, Friedman,
2004). These methods are convenient but lack accuracy and the amount of kernel flour needed was around 50g which is sometimes unfeasible in the steps of a 29 breeding conversion. Currently, with application of UPLC, much smaller quantities
(1g of flour or single kernel) are sufficient for amino acid profiling which facilitates breeding without excessive usage of samples for destructive analysis.
2.3 Comparison of o2 and QPM with wild type
Advantages of o2 mutants
Several feeding trials and nutritional studies were carried out to verify the superior
protein quality of o2 mutants (both unmodified o2 and QPM). Nutritional value of o2 corn, high protein o2 corn and normal corn were compared (Gipp and Cline,
1972). On an equal weight basis with normal corn in feeding trials of rats, the average daily gain of o2 corn and high protein o2 corn was almost five times of those obtained with the normal corn diet (5.20 g and 5.94g compared with 1.22g).
The nutritional benefits of QPM were confirmed in feeding trials (Mbuya et al., 2011;
Panda et al., 2010; Sofi et al., 2009; Krivanek et al., 2007). Other feeding trials were
carried out (Bressani, 1966; Mertz et al., 1965; Pickett, 1966; Cromwell et al., 1969;
Jensen et al., 1969). In a feeding trial of weaning pigs, an optimized diet of QPM was
suggested (72.3% inclusion rate of QPM in pig weaner diets) with higher efficiency in
food conversion and at a reduced cost of production (Mpofu et al., 2012). The effectiveness of o2 in its increased nutrition was confirmed in feeding weaning rats
(Nelson et al., 1965).
Disadvantages and adverse effects of o2 mutation
The kernels of o2 mutants are of poor and unacceptable quality due to their soft,
low density kernels. Disadvantages of o2 maize include the following aspects: 30 starch granules loosely packed (soft kernel), low kernel weight, high kernel
breakage, low grain yield, chalky and soft endosperm resulting in damaged kernel
while harvesting, poor germination, increased susceptibility to pest and diseases,
and inferior for food processing. Previously, lower grain yield was observed on o2 plants; o2 kernels weigh 10 to 15% less than normal kernels (Alexander, 1966). o2 inbreds and hybrid plants generally yield less than their normal counterparts
(Alexander, 1966; Nelson, 1967; Salamini and Ekpenyong, 1967). Data collected supported the disadvantages of o2 in hybrid performance included grain yield (in
quintals), moisture percent of grain at harvest, percent of erect plants, 100 kernel
weight, percentage of cracked kernels, oil percentage, protein percentage in maize grain
and lysine content. Compared with normal hybrids, o2 hybrids were significantly lower
in grain yield (8%), weight per 100 kernels (5% lower), but higher in grain moisture
(20%), percentage of cracked kernels (89%), oil content (13%), protein (2%) and 52%
more lysine on average (Lambert et al., 1969).
The O2 gene mainly affects kernel characters and phenotypic variability is mainly related with genetic causes implying that selection for suitable hybrid combinations might result in amelioration of the negative effect of o2 (Salamini et
al., 1970; Lambert et al., 1969). Comparison of o2 and normal inbred lines in terms
of resistance to ear-rotting pathogens revealed that o2 were less resistant to ear-rot damage than the normal versions (Loesch et al., 1976). The o2 mutation causes numerous pleiotropic effects in plants but also biochemical effects in the grain
(Giroux et al., 1994; Yunes et al., 1994), and also in leaves (Morot-Gaudry et al.,
1979). Adverse effects of o2 germplasms included reduction in yield (Salamini et al., 31 1970). Early introgression of o2 into inbred lines of high-yielding hybrids led to a
substantial 10% or more reduction of yield (Crow and Kermicle., 2002). Fungal
infection was problematic in o2 kernels drying slowly (Crow and Kermicle, 2002).
3 Mechanism of amino acids change and protein quality improvement
o2 mutants showed different amino acid profile compared to the counterpart of
wildtype. Several studies confirmed the effect of o2 in amino acid difference.
3.1 Mechanism of amino acid changes in o2
3.1.1 Pathway for lysine synthesis and degradation
Lysine and tryptophan are first and second limiting amino acids respectively in
maize (Bright et al., 1983). Genetic control of lysine accumulation in maize endosperm was related to the rate of lysine conversion (Da Silva and Arruda,
1979). Monofunctional aspartate kinase genes (Ask1 and Ask2) in maize were
characterized and it was found that the enzyme encoded by Ask2 in Oh545o2 bears greater total activity than that encoded in Oh51Ao2. This might result in higher level of free amino acids (Wang et al., 2007).
Mutations increasing lysine content in the endosperm were demonstrated both in
maize (Sodek and Wilson., 1970) and in barley (Brandt, 1975). Identification of lysine-containing proteins was carried out and subsequent sequence analysis revealed that the corresponding mRNAs are predicted to be involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis (Habben
et al., 1993). In silico dissection of the the most significantly changed proteins in o2, 32 fl2 mutants compared with wild type suggested that protein compositional changes
may contribute to the difference in lysine content (Morton et al., 2015). The enzymes
involved in lysine biosynthesis are plastid localized (Figure 6) (Bryan, 1990; Galili,
1995). Enzymatic catabolism of lysine in seed was studied (Figure 7) (Galili, 1995).
Expression of the dihydrodipicolinate synthase from E.coli in tobacco confirmed
the significant correlation between content of free lysine and level pf DHDPS
activity (Kwon et al., 1995). In higher plants, lysine is synthesized from aspartate in one branch of the aspartate family pathway, similar to many bacterial species
(Galili, 1995). AK (aspartate kinase) is the first enzyme in this pathway whose
activity can be inhibited by lysine (Galili, 1995). Elongation factor 1 α (eEF1α1) concentration was confirmed to be highly correlated with lysine content of maize endosperm (Habben et al., 1995). The roles of lysine residue in proteins were reported
as is related to receptor affinity, endoplasmic reticulum retention, protease cleavage
points, enzyme catalysis, nuclear structure and function and muscle function (Flodin,
1997). Aspartate is the common starting molecule for synthesis of lysine, threonine, methionine and isoleucine in higher plants (Azevedo et al., 1997). The
dihydrodipicolinate synthase was confirmed to be expressed in a cell specific manner in Arabidopsis (Vauterin et al., 1999). Enzymes involved in lysine metabolism were compared across wild type, o2 maize and QPM. QPM exhibited higher levels of aspartate kinase activity and most reduced activity of lysine ketoglutarate reductase saccharopine dehydrogenase compared with that of wild type and o2 (Gaziola et al., 1999). Seed-specific expression of a bacterial lysine
feedback-insensitive DHDPS resulted in up to a 100-fold increase in free lysine 33 accumulation in seeds of transgenic Arabidopsis, canola and soybean (Falco et al.,
1995; Zhu and Galili, 2003). In contrast, significant accumulation of lysine in maize
seed could only be achieved through deregulating lysine anabolism in the embryo
(Mazur et al., 1999; Huang et al., 2005). Influence of lysine biosynthesis on pathways for methionine and threonine was discussed in Arabidopsis (Craciun et al., 2000). It
was suggested that the DAP pathway is related to arginine metabolism and AAA pathway is related to Leu pathways (Velasco et al., 2002). The lysine biosynthesis pathway has two different anabolic routes, one is the diaminopimelic acid pathway and the other one is the α-aminoadipic acid route (Velasco et al., 2002). Monsanto released the first genetically modified (GM) high lysine corn which was developed by expressing the feedback-insensitive DHDPS gene from Corynebacterium (Shewry,
2007). High-lysine corn was generated by suppression of lysine catabolism (via suppression of the lysine catabolic enzyme LKR/SDH) in the endosperm via RNAi
(Houmard et al., 2007). Protein composition and lysine content of opaque mutants (o1, o2, o5, sh4-o9, o11, Mc, DeB30, fl2, fl1, fl3) was summarized (Shewry, 2007). Lysine, threonine and methionine are synthesized from aspartic acid (Shewry, 2007).
DHPS (the Dihydrodipicolinate Synthase) is the enzyme that catalyzes the first
reaction that is unique to lysine biosynthesis. This enzyme was proven to be strongly inhibited by lysine (Varisi et al., 2007). The higher concentrations of
lysine observed in mutant kernels may be explained by the reduction of lysine
degradation or by changes in the distribution of high lysine containing storage
proteins (Varisi et al., 2007). In a study, tryptophan was monitored because of its simple and fast determination method in a QPM breeding program (Ignjatovic-Micic 34 et al., 2010).
Bacterial lysine biosynthesis-related enzymes were studied including DHDPS
(dihydrodipicolinate synthase), DHDP (dihydrodipicolinate) and DHDPR
(dihydrodipicolinate reductase) (Dogovski et al., 2012). Lysine-rich proteins were identified and characterized in o2 mutants and might contribute substantially to lysine increase of o2 endosperm (Jia et al., 2013). For example, increased
accumulation of GAPDH and SDH1 which contains over 8% and 4% lysine respectively was observed in W64Ao2. Increased content of Asp and Asp derived amino acids (lysine ethionine and threonine) were reported in the loss of function mutation in the AK-HSDH2 (Asp kinase-homoserine dehydrogenases) gene in
Arabidopsis (Teresa and Lu, 2015). The increase in non-zein proteins may be a
response to an increase in available amino acids (Sodek and Wilson, 1970). Such
protein changes in o2 germplasms compared with wild type, including the reduced zein
content and increased non-zein content, referred to as proteomic rebalancing, result in
increased content of two essential amino acids (Lysine and Trptophan). A comparison was made between o2 and normal maize via the utilization of isotopically labeled carbon element to lysine and leucine confirming that Lysine is absent from zein, whereas leucine is exceptionally high (Sodek and Wilson, 1970). During endosperm
development, carbon was incorporated into endosperm proteins. It was found that
injected isotope-labelled lysine was converted to glutamic acids and proline in normal
maize endosperm while little conversion was observed in o2 endosperm, most of the
isotope-labelled carbon was found in lysine, whereas in the normal endosperm the
isotope-labelled carbon was found in glutamic acid, proline and lysine. It was reported 35 that aspartate semialdehyde dehydrogenase was not feedback-inhibited by lysine
(Gengenbach et al., 1978). Although some lysine synthesis may occur in the
endosperm (Sodek, 1976), lysine may be largely synthesized in other tissues and
then transported to the kernels (Da Silva and Arruda, 1979). The activity of lysine- ketoglultarate reductase was studied and was thought to be associated with lysine breakdown in the endosperm during seed development (da Silva, 1983). Partial
purification and characterization of lysine-ketoglutarate reductase was carried out in both normal and o2 maize endosperms (Brochetto-Braga et al., 1992).
LKR/SDH activity, both involved in lysine degradation in maize endosperm,
coincides with storage protein synthesis and accumulation, and is strongly
depressed several fold in o2 mutants as compared to wild type (Azevedo et al.,
2003; Brochetto-Braga et al., 1992). Lysine derived from aspartate in the pathway that consists of several strongly regulated enzymes (Azevedo et al., 1997). The
first enzyme unique to lysine synthesis, dihydrodipicolinate synthase (DHDPS; EC
4.2.1.52) has also been extensively studied and characterized in plants catalyzing
the condensation of pyruvate and aspartate semialdehyde into dihydrodipicolinic
acid (Azevedo and Lea, 2001). DHDPS is also subject to feedback inhibition by
micromolar concentrations of lysine (Azevedo and Lea, 2001). The two key enzymes
in the lysine biosynthetic pathway aspartakinase (AK) and dihydrodipicolinic acid
synthase (DHDPS) are feedback inhibited by lysine (Galili, 2002). Expressing both
sense and antisense 19-kDa α-zein genes were used to improve nutritional quality of maize proteins (Huang et al., 2004). Reduction of α-zein via transgenic venue was confirmed to result in high lysine and high tryptophan (Huang et al., 2006). The 36 o2 mutation reduces the production of enzyme lysine keto-glutarate reductase,
resulting in a free-lysine increase (Xu et al., 2008). The O2 gene controls the
transcription of the gene encoding the bifunctional enzyme LKR/SDH (lysine-
ketoglutarate reductase/saccharopine dehydrogenase), which is the first enzyme of
lysine catabolism (Prioul et al., 2008). In a study comparing two types of zein reduced kernels with wild type, increased lysine was confirmed to be mainly because protein-bound lysine increase which is different from the situation in o2 kernels that free lysine contributed partially to the increased lysine because of the reduced production of lysine keto-glutarate reductase (Frizzi et al., 2010). The aspartate pathway includes synthesis of lysine, threonine, methionine and S-
adenosylmethionine (Azevedo et al., 1997). In a study on three enzymes of the aspartate pathway which can lead to production of lysine, theronine and methionine, only the first two enzymes, which are aspartate kinase and homoserine dehydrogenase) have reduced activity compared with those of wild type (Azevedo et al., 2003). An pathway, the α-aminoadipate pathway which is unique to fungi
was discovered (Xu et al., 2006). The lysine biosynthesis pathway is not fully
understood. Genes involved in lysine biosynthesis pathway (LBPG) in B73 were
identified (Liu et al., 2016). Genes for key enzymes in this pathway,
dihydrodipicolinate synthase, were analyzed for contribution to lysine synthesis
(Liu et al., 2016). Coexpression network of LBPGs revealed that genes which
coexpressed with LBPGs encode ribosomal proteins, elongation factors and zein
proteins. The conversion of lysine into glutamic acid and proline was very high
compared to the conversion into all other compounds (Sodek and Wilson, 1970). 37 However, while lysine is increased in the o2 background, while comparing o2 and
wildtype, glux reduction was found. Nigam et al observed that in wheat seedlings
injection of isotope-labelled lysine results in glutamic acids (Nigam and
McConnell, 1963). Lysine pathway involves pipecolic, aminoadipic and glutaric
acids (Nigam and McConnell, 1963).
3.1.2 Biosythesis of other amino acids
Same with lysine, Trp (tryptophan) and Met (methionine) are also limiting for human beings. For Met content, a maize inbred line BSSS53 was characterized with an elevated content of grain methionine (Phillips et al., 1981) which was later confirmed be the result of elevated 10-kD δ-zein content in the endosperm
(Phillips and McClure, 1985; Phillips et al., 1981). The methionine content in maize kernels is largely determined by the level of methionine rich 10-kDa protein which is encoded by the gene Zps 10/(22) (Schickler et al., 1993). HSD
(Homoserine dehydrogenase) catalyzes the first reaction that is uniquely associated with methionine biosynthesis (Galili, 1995). Methionine was increased in maize seed when the cis-acting site of the Dzs10 gene, which encodes a seed- specific methionine storage protein (10kD δ-zein), was replaced (Lai and Messing,
2002). Expression of high methionine proteins such as 2S albumins and SFA8 can result in methionine increase (Shewry, 2007). To increase methionine in maize, δ- zein (which contain 23% or more methionine) has good potential as an endogenous protein. Reducing the endogenous sulfur-poor proteins might also improve the methionine content (Holding and Larkins, 2008). Two genes (dzs 10, 38 dzs18) in the maize endosperm were proven to have high percentage of methionine
codons (Wu et al., 2009). Methionine-rich zeins (such as 10 kD δ- zein and 15-kD β- zein) were increased in seed when leaf serine acetyltransferase was transformed and overexpressed (Xiang et al., 2018).
3.2 Protein quality improvement
Strategies for lysine improvement include the following three categories (Bright et al., 1983) 1. low prolamin mutants; 2. increases in specific lysine rich proteins; 3.
changes in free amino acids. These categories are also categorized as naturally
occurring or induced mutations and transgenic.
3.2.1 Conventional approach for protein quality improvement
The principal efforts in improvement of protein quality in cereals have been
directed towards incorporation of naturally occurring or induced mutant genes
(Bright et al., 1983). One of the two is introgression of o2 to increase the amount
of two essential amino acids lysine and tryptophan. Avenues for the aim of lysine
increase include increasing lysine-rich proteins and decreasing zein synthesis
(Shewry, 2007). By combining two strategies which are reduction of zeins and
expressing lysine biosynthetic enzyme, researchers successfully increased total
lysine content to nearly double the amount in F1 progeny (Huang et al., 2005).
3.2.1.1 Marker-assisted breeding for gene introgression
Definition and principles
Marker-assisted introgression (also known as marker-assisted selection (MAS), 39 marker-assisted breeding) is defined as the transfer of single or multiple genes from
donor genotypes conferring traits of interest into target germplasms by repeated backcrossing. Requirements for marker-assisted breeding were summarized
(Stam, 2003). Marker-assisted breeding is based on the accurate associations between
traits and markers and the effectiveness is related to factors including population sizes,
number of selection markers in relation to chromosome number and genomic map
length (Stam, 2003). Markers were used to hasten the recovery of the recipient genome in introgression breeding program (Hospital et al., 1992). The advent of
various type of markers including restriction fragment length polymorphism
(RFLP), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) has greatly facilitated breeding programmes (Jonah et al., 2011).
Simulation studies of different breeding schemes
With marker information such as linkage distance and marker density in hand,
computer-based simulation can be carried out for estimation of the efficiency of
marker-assisted breeding. Simulation of marker-assisted introgression of quantitative
trait loci concluded that at least three markers per QTL allows good control over
generations (Hospital et al., 1997). The efficiency of gene introgression via
backcrossing was simulated and strategies applying selections at different
backcrossing generations were compared (Frisch et al., 1999; Ribaut et al., 2002).
Factors affecting the success of marker-assisted selection include number of target genes to be transferred, distance between the flanking markers and the target genes, 40 number of genotypes selected in each breeding generation, the nature of
germplasm and the technical options available at the marker level (Gopalakrishnan
et al., 2008). Considerations for introgression of exotic germplasm (one gene or
two independent genes) into F2, BC1 and BC2 populations, were listed while taking
population sizes, gene frequencies and intensities of selection into account (Crossa,
1989). Frisch compared the selection strategies for marker-assisted introgression of a gene via computer simulation and concluded that substantial savings can be achieved by venues including increasing population size, selection of recombinants for target allele in early generations (Frisch et al., 1999).
The application of MAS has facilitated a variety of crop breeding projects
including barley, wheat, rice, soybean and maize. MAS was carried out in barley
for Yd2 gene conferring resistance to barley yellow dwarf virus (Jefferies et al.,
2003). A gene conferring high grain protein content was introgressed into bread wheat cultivars via MAS (Kumar et al., 2011). Foreground and background selection were integrated together in selection and identification of superior
Basmati rice which is resistant for bacterial blight (Gopalakrishnan et al., 2008).
Marker-assisted selection was successfully used for pyramiding resistant genes in soybean (Maroof et al., 2008). Babu et al proposed that combination of foreground selection (for the o2 mutation), background selection (for recurrent genome) and
phenotypic selection for quantitative traits would be effective in trait introgression projects (Babu et al., 2004).
Besides introgression of one gene of interest, to incorporate more genes into certain 41 genetic background, gene pyramiding was adopted. Marker-assisted gene
pyramiding was aimed at assembling multiple desirable genes into a single
genotype (Ye and Smith, 2008). Genes waxy and o16 were successfully pyramided via marker-assisted breeding in a cross and backcross breeding scheme (Yang et al.,
2013).
For the success of its application, MAS requires several prerequisites. For QTLs of
traits of interest, it is important that QTLs are precisely mapped and validated before
application for trait introgression (Ye and Smith, 2008). Difficulties arose for QTL
pyramiding including less precision resulting in linkage drag, efforts in validation of
QTLs effect in the target genetic background, and the fact that QTL expression might
be environment-specific (Ye and Smith, 2008).
3.2.1.2 QPM conversion studies
Incorporation of o2 gene into a high total protein strain was carried out (Nelson, 1966).
QPM conversion is one avenue among several other types for maize biofortification.
The o2 allele and QTLs were combined to generate elite germplasms with hard kernel and increased nutrition (Vasal et al., 1980). Introgression of o2 into elite inbred lines
(CML202 and CML204) which are resistant to imidazolinone herbicides was
carried out (Danson et al., 2006). Seven individuals were selected from the BC3F3
population as QPM inbred lines from a MAS program for introgression of the o2
gene (Magulama and Sales, 2009). Introgression of o2 and o16 genes successfully increased lysine content by up to 94.3% in waxy maize backgrounds (Zhang et al.,
2013). One commercial inbred line from Maize Research Institute Zemun Polje 42 (MRI) was successfully converted to QPM version in the BC2F3 families
(Kostadinovic et al., 2014). To make QPM lines adapted to temperate regions,
QPM conversion was carried out for ZPL3 and ZPL5 which are elite lines for temperate regions (Kostadinovic, 2015). o2 was introgressed into waxy maize line
Zhao OP-6 (O2O2) by MAS resulting in improved lysine (increased by 51.6%)
(Zhou et al., 2016). It was proposed for QPM development that continuous
monitoring of lysine and/or tryptophan content is required instead of only at the
final step (Vivek et al., 2008).
3.2.2 Application of transgenic approaches for protein quality improvement
Genetic transformation is defined as the process of delivering isolated or modified single genes into other species via molecular cloning and genetic transformation techniques. (Bright et al., 1983). Transgenic expression of seed storage proteins can be applied for improvement of seed and nonseed crops (Holding and Larkins,
2008). Advantages of genetic transformation include that this eliminates the issue of retention of unwanted and genetically-linked traits (Ye and Smith, 2008). Efforts on modification of zein genes to contain lysine and tryptophan showed successful synthesis of zein proteins which did not interfere with their association into protein bodies in the Xenopus laevis oocytes, suggesting the potential of zein modification in transgenic maize systems (Larkins et al., 1993). A transgenic approach was applied to circumvent feedback-regulation of two enzymes of the lysine biosynthesis pathway, AK and DHDPS, resulting in increased lysine content in canola 43 and soybean (Falco et al., 1995). RNA interference (RNAi) was carried out in order to
silence 22-kDa α-zein resulting in increased lysine content (Segal et al., 2003), and 19-
kDa α-zeins respectively (Huang et al., 2004; Huang et al., 2005). Wu et al adopted
RNA interference (RNAi) to reduce 22-kDa and 19-kDa zeins resulting in
increased lysine (Wu and Messing, 2011).
4. Popcorn
Popcorn is characterized as having a high proportion of vitreous endosperm. Several
physical properties of popcorn were studied including kernel length, width, thickness,
arithmetic mean diameter, and geometric mean diameter, sphericity, kernel volume, kernel surface area, thousand seed weight, true density, bulk density, static
coefficient of friction and dynamic angle of repose (the steepest angle of descent or dip relative to the horizontal plane to which certain popcorn seeds can be piled without slumping) (Karababa, 2006). Several benefits associated with popcorn were reported.
Popcorn is included among foods suggested in the Dietary Guidelines For Americans
(2005) and MyPyramid (Center for Nutrition Policy and Promotion) to increase whole
grain consumption ( Grandjean et al., 2008). From a study comparing the dietary data of popcorn consumers and non-consumers among Americans, popcorn consumption was associated with increased intake of whole grains, dietary fiber and certain other nutrients (Grandjean et al., 2008). Twenty-three odorants were detected in popcorn
(Schieberle, 1991) including 2-acetyl-1-pyrroline, (E,E)-2,4-decadienal, 2- furfurylthiol, and 4-inyl-2-metho-xyphenol which showed the highest odor activities
(Schieberle, 1991). Composition analysis of different popcorn types (depending on 44 how the appendages were expanded, unilaterally, bilaterally or multilaterally) revealed
that unilateral popcorn flakes retained endogenously the most total fat, saturated fat,
and sodium whereas multi-lateral type had the highest levels of endogenous protein,
total carbohydrate and aromatic pyrazines (Sweley et al., 2011).
4.1 Types of popcorn
Based on the shape of the popped kernels, popcorn can be divieded into two
categories, butterfly with an irregular, branched or prolonged appearance and
mushroom with a round shape (Eldredge and Thomas, 1959). Mushroom type was preferred for confection purpose because of ease in caramel or sugar coating and also in handling and preventon of flake breakage after packaging. Based on the shape of kernels, popcorn can be divided into two groups pearl and rice (Eldredge
and Thomas, 1959). The pearl type includes round-shaped kernels. The rice type is characterized with a sharp pointed crown. Different popcorn flakes (unilateral,
bilateral, multilateral) were identified from a yellow butterfly popcorn hybrid (YP-
213) (Sweley et al., 2011). Common popcorn germplasms that are of commercial importance include White Rice, Jap Hulless (Japanese Hulless), and Queen Golden
(Brunson et al., 1937). A mushroom type, named South American, became a major
competitor of the other varieties in the early 1900s. Supergold, also known as
Sunburst or Yellow Pearl, were also of commercial importance.
4.2 Popcorn quality
Compared with normal dent maize germplasm, popcorn varieties have agronomic
deficiencies. Normal maize germplasm (dent corn) might facilitate yield 45 improvement of popcorn. Li et al studied genetic regions associated with grain
yield variation between dent corn inbred Dan232 and popcorn N04 (Li et al.,
2007). Popcorn normally lacks strength of stalk and resistance to root lodging.
These traits can be introduced from dent corn by crossing. Crosses were made between popcorn and dent corn parents to study the inheritance of popping related characteristics (Crumbaker et al., 1949). The term lodging, also called root lodging, refers to the tendency of some hybrids to lean over which is different from breakage above the ground which is called stalk lodging (Eldredge and Thomas,
1959).
4.3 Popping process and mechanism for popping
The major trait that separates popcorn from all other types of corn is the formation
of expanded flakes after the kernel explodes in response of heating. This trait is
referred to as popping or kernel expansion. Popping expansion volume is of utmost
importance because popcorn is often sold in its popped form by volume (D’Croz-
Mason and Waldren, 1978). The popping process is not one explosion but a chain
reaction of millions of tiny explosions as each starch grain expands and bursts.
Popping is a simultaneous starch gelatinization and expansion process of kernels
while being heated with high temperature for a short time (Mishra et al., 2014).
The factors conditioning popping include optimum moisture content, an elastic and
tenacious confining structure and proper application of heat. Kernel moisture provides
a source of steam while heating. Popping ability is not restricted to popcorn. Some
hard flinty grain sorghums also pop well. Some hard shiny seeds of pigweed can 46 also pop (Eldredge and Thomas, 1959). Popping characteristics can be found in dent corn, flint corn and sorghum (such as Pink kafir) but is exhibited the most in popcorn (Zea mays everta) (Brunson et al., 1937). The popping sound was confirmed to be caused by the release of water vapor (Virot and Ponomarenko,
2015).
Comparison between popped and unpopped kernels
Structural differences of the popped and unpopped grain were observed by Parker et al
(Parkeret al., 1999). When unpopped grain was fractured, polygonal starch granules surrounded by protein bodies were tightly packed in the endosperm of the grain.
However, air spaces were observed in the counterpart of popped grain.
Scales for evaluation of popping expansion (Popping Expansion Volume)
A weight volume test is used to measure popping volume. A weight volume test is usually carried out by measuring the weight of popping samples and recording the popped samples (Eldredge and Thomas., 1959). It was discovered that low popping volume was partially dominant over high popping volume in dent x popcorn
crosses (Crumbaker et al.,1949). An arbitrary scale was used to evaluate popping
expansion which ranges from 1 to 5 with 1 = kernels does not pop; 2= pericarp bursts; 3= kernel pops, endosperm puffs slightly; 4= kernel approaches normal popping but with distinctly subnormal expansion; 5= normal popping.
Factors affecting popping volume
Good popping quality is influenced by two factors which are kernel expansion 47 (volume increase) and crispness of the popped kernels (Station and Duncan, 1934).
Factors to be used are as follows: 1. The moisture content of the ears should remain
as constant. 2. Popping conditions such as degree of heat, popping instrument
would also affect the popping process. 3. Routine procedure in measuring the
volume. Comparisons were made between popcorn coated with zein and untreated
popcorn during the popping process and an increase of 15% was found in expansion
bulk volumes for coated popcorn (Wu and Schwartzberg, 1992). Factors including
salt, pericarp thickness, kernel volume sphericity, diameter ratio, thousand kernel
weight, hardness, and density were investigated for their effects on popping
volume over 18 popcorn hybrids where pericarp thickness is highly correlated with
expansion volume (correlation being 0.729) and salt had a negative effect on
popping volume (Mohamed et al., 1993). The relationship between lipids starch and storage protein composition and popcorn expansion was studied (Borras et al.,
2006). Characteristics including flake size, 1000-kernel weight, kernel size and percentage of unpopped kernels, protein content were reported to have direct positive effect on expansion volume (Soylu and Tekkanat, 2007). Right popping
techniques of popping and puffing include sand roasting, salt roasting, gun puffing,
hot oil frying, and heating via medium (hot air or microwave radiation) (Jaybhaye et
al., 2014). Eleven commercial popcorn hybrids, one flint corn x popcorn cross and one
open pollinated popcorn variety performed better using conventional popping than
using microwave popping (Dofing et al., 1990).
Different factors can affect popping results. For application of different oil, significant negative correlation (R = –0.702; P < 0.005) was observed between 48 endogenous oleic acid and popping expansion whereas a positive correlation (R =
0.612; P< 0.02) was observed between endogenous linoleic acid and popping
volume (Borras et al., 2006). Also, over different expansion times, comparison was
made between popping result with addition of oil and without addition of oil and
it was found that addition of oil can accelerate the gelatinization process of starch
(Paraginski et al., 2017). To study the temperature effect, kinetic behavior for
popping kernels were recorded under two different treatment (oil or air) where
minimum popping temperature were predicted respectively as 181± 2°(oil) and
187± 2°(air) (Byrd and Perona, 2005). Percentage of soft starch increases with size of kernel and per cent expansion decreases as size of kernel increases (Station and
Duncan, 1934). It was observed that smallest-sized kernels have better popping properties than larger fractions and smallest kernels have the highest expansion volume (Ceylan and Karababa, 2002). In a study assessing popping ability of new tropical hybrids, a significant negative relationship was observed between flake volume and kernel size (r=-0.27) while number of unpopped kernels was positively correlated with kernels size (r=0.56) (Jele et al., 2014). Popping volume and number of unpopped kernels is significantly affected by kernel size and genotype
(Song et al., 1991). About 12-14 percent moisture gives maximum popping volume
(Brunson et al., 1937; Station and Duncan, 1934). Increased popping volume was produced by decreasing the pressure surrounding the popcorn kernels (Arkhipov et al., 2005).
Starch is also an important factor in the popping process. The popping properties of the various types of corn follow closely the relative proportions of hard or 49 vitreous starch in the endosperm (Willier and Brunson, 1927). Brunson et al
studied how starch grains are embedded in a colloidal material, resulting in
confined room for increased steam pressure which is required for popping
(Brunson et al., 1937). The theory behind popping expansion is that the thickness
or toughness of the protein matrix surrounding the starch grains holds in the
moisture until sufficient steam pressure is generated to cause an explosion
(Eldredge and Thomas, 1959). It was found that popcorn starch properties were
significantly different from normal corn whereas no particular measured attribute
of starch including amylose and amylopectin content correlated with popping
volume (Borras et al., 2006). Comparison of F1, BC1 and BC2 in terms of kernel starchiness and popping volume showed a significant correlation (ranging from
0.34 to 0.76) between popping volume and kernel starchiness (Crumbaker et al.,
1949). Factors affecting popping result include moisture level, seasoning (oil or fat applied) and temperature (Eldredge and Thomas, 1959). The ideal moisture content for popping is between 13 and 14 percent. Depending on the amount of popcorn popped at one time, temperature for best popping was reported to range from 350°F to 530°F (Eldredge and Thomas, 1959). To achieve high expansion of popcorn, complete maturity of kernel and a dense and elastic endosperm is required
(Brunson et al., 1937). Properties of the endosperm can also affect result of popping expansion, which includes seed characteristics such as proportion of soft
(opaque) starch and size of kernel. Popcorn contains very little soft starch in comparison to most dent corns. Floury types of corn do not pop because the kernels are made up entirely of soft starch (Eldredge and Thomas, 1959). Proportion, 50 distribution and location of the hard and soft starch in the endosperm varies in
popcorns with different popping volumes. The per cent of soft starch is relatively small in the higher expansion strains and it’s located in the interior of the endosperm in close proximity to the germ (Station and Duncan, 1934). Factors
conferring excess soft starch in popcorn includes immaturity, cross contamination
from dent, flint or flour corn (Station and Duncan, 1934). Several characters were studied including expansion volumes, percentage of soft starch weight of 100 kernels, number of kernels in 25 cubic centimeters and length of kernels breadth of kernels and thickness of kernels (Willier and Brunson, 1927). The results showed that there is good correlation (r=-0.59±0.022) between the amount of soft
starch in the center of the kernel and the popping expansion of the ear where it
came from (Willier and Brunson, 1927). The negative value of the correlation
coefficient means that kernels with the least amount of soft starch in the kernels
have the best popping potential. Furthermore, it was found that large kernels (as
indicated by weight of 100 kernels) gave lower popping volume than small ones
with a correlation coefficient r= -0.31 and large kernels tend to have high percent
of soft starch (r=0.42) (Crumbaker et al., 1949). Instead of gelatinization during
the popping process in the translucent endosperms of popcorn, the starch granules
in opaque endosperms do not get penetrated by water vapor (Sweley et al., 2013).
Beside the factors mentioned above, popping expansion volume can be influenced by
certain genes. To see the effect certain genes might have on popping, 14 endosperm
genes (including shrunken-1, shrunken-2, brittle-1, sugary-1, sugary-2, amylose extender, dull, shrunken floury, opaque1, opaque2, floury-1, floury-2, waxy and 51 soft starch) from dent corn backgrounds were crossed to popcorn lines (Richardson,
1959). As expected, genes conferring a floury textured endosperm (o1, o2, fl1, fl2)
inhibited normal expansion because of the inverse relationship between proportion
of soft starch and popping expansion. From this finding, we can expect that starchy
endosperm would result in less popping volume.
4.4 Popcorn breeding for incorporation of desirable traits
Yield
Yield and popping expansion were selected across seventy five full-sib popcorn families and analysis of variance indicated sufficient genetic variability for further
selection (Daros et al., 2002). The trait affecting yield the most in popcorn is the number of seeds per row on popcorn ears (Thomas and Grissom, 1961).
Quality traits
Grain yield and quality traits of 18 popcorn hybrids and four commercial popcorn
cultivars were determined (Öz and Kapar, 2011). Combining ability was estimated
between eight tropical popcorn lines and one temperate popcorn line in terms of
seed quality related traits and popping related traits (height of the first ear insertion,
grain yield and popping expansion) (Moterle et al., 2012). Improvement of popcorn
focused on quality traits including popping volume, shape of popped kernel,
tenderness and flavor (Crumbaker et al., 1949). A colored popcorn variety was
developed with high anthocyanins (66mg/100g in unpopped ground kernel
powder) which confers marked antioxidant capacity compared with colorless
control (Lago et al., 2013). Crumbaker et al studied the segregation of kernel 52 starchiness and indentation among BC1S1 population (Crumbaker et al., 1949). The
distribution of kernel indentation was observed in BC1 populations. The dent characteristics of the ears were partially dominant over the flinty character of popcorn (Crumbaker et al., 1949). Besides the desirable characters to be achieved for dent corn breeding, popping expansion and tenderness of popped kernels are of great importance which requires paramount consideration. Also, after popping, absence of coarse hull and presence of good flavor are also desirable. There is
difficulty in combining top yields and superior popping expansion. High yield also
produces too much soft starch in the centers of the kernels (Brunson et al., 1937).
This results in compromise being made in either yield or popping expansion. For
popcorn selection, any ears that have smutted cracked discolored kernels or kernels which show any indications of excess soft starch should be discarded (Station and
Duncan, 1934).
Mapping of PEV
QTLs for PEV were mapped on chromosomes 1, 3, 8, and 10 using 194 F3 families derived from a cross between a popcorn (A-1-6) and a flint corn (V273) (Babu et al.,
2006). For the use of dent corn as parents in crosses to add trait to popcorn, successful
recovery of popcorn quality trait is essential. To this end, backcrossing strategies using
popcorn as recurrent parent can be adopted to recovery popcorn quality trait. It was
proved that popping volume may be sufficiently well-recovered to make possible
improvement of popcorn lines by out-crossing to dent corn inbreds, suggesting that the
mechanism of inheritance for popping volume is comparatively simple (Johnson and 53 Eldredge, 1953). BC2 population from a dent x popcorn cross has three-fold higher
popping volume compared with parental popcorn lines (Crumbaker et al., 1949). This indicates that popping volume can be recovered and two times of continuous backcrossing seemed sufficient. In addition, increased size of ear was observed in BC2 compared with popcorn inbred (almost double in terms of average size) (Crumbaker et al., 1949). Because of the importance of popping volume and quality in the utilization of popcorn, improvement of popcorn by introducing trait of interest from dent corn would require backcrossing with popcorn parental lines as recurrent parent (Johnson and Eldredge, 1953). High popping volume can be recovered in F2 and backcross
populations (Glisson, 1951). In a study to introduce lodging resistance, recovered lines
in BC1S4 and BC2S4 originated from a dent (W22) x popcorn cross also showed
essentially equal popping volume (Johnson and Eldredge, 1953). BC1S4 has wider range of popping volume than BC2S4. Ears with high percentage of kernels of similar visual appearance to that of the popcorn parent showed high mean popping volume
(Crumbaker et al., 1949).
Cross-incompatibility
Cross-incompatibility, also known as non-reciprocal cross-sterility, was defined as a
phenomenon that certain maize materials that won’t produce seeds when pollinated
with pollen from other maize, whereas its pollen can be used to pollinate silks of other
maize plants, resulting in successful fertilization (Zeng, 1998). The first recorded
observation of cross-incompatibility was on F2 of a popcorn x sweetcorn cross by
Correns in 1902 (Correns, 1902). Demerec et al found popcorn ovules were rarely 54 fertilized by dent corn gametes when pollinated by pollen from dent corn in 1929
(Demerec, 1929). Incompatibility was also reported between South American and
Supergold varieties by Brundson et al in 1937 and in 1945 respectively (Brunson, 1937;
Brunson and Smith, 1945). Nelson later found this non-reciprocal cross- incompatibility to be widespread among many popcorns of diverse varietal origin in
1952 (Nelson, 1952). The reason it is common in specific maize germplasms is still not clear and requires further studies of phylogenetics using homologous sequences of characterized Ga gene. The genes underlying this phenomenon were known as gametophyte factors (Ga). Cross-incompatibility usually is conferred by one major gene and there are also some minor modifier genes (Zhao et al., 2014).
The cross-incompatibility systems have male function and female function. The male function enables pollen to overcome the incompatible barrier. The female function (also known as pistil barrier function) prevents pollen of different genotype from silk itself from successful pollination. There are three allelic forms (the strongest allele, Ga1-s; the allele containing only the male function, Ga1-m; the cross-fertile allele accepting pollen from all allelic forms, ga1) of gametophytic factors based on the completeness of cross- incompatibility. The relationship between the three different allelic forms of Ga locus is shown in Figure 8 (Jones et al, 2015). Ga1-s is one form of the alleles at Ga locus and the relationship between Ga1-s/Ga1-s and ga1/ga1 is complete non-reciprocal cross- incompatibility (or unilateral cross-incompatibility). From Figure 8, we can see female plants homozygous for Ga1-s/Ga1-s will end up with completely failed fertilization after pollination by pollen carrying the ga1 allele, whereas pollen carrying Ga1-s allele will successfully fertilize female plants homozygous for ga/ga or Ga1-s/Ga1-s. Compared with 55 Ga1-s, Ga1-m has only the male function but lacks the female function. We can see male plants homozygous for Ga1-m/Ga1-m will pollinate ga1/ga1 and Ga1-s/Ga1-s females successfully but will fail to create pistil barrier when pollinated by Ga1-s/Ga1-s and ga1/ga1 males. Ga1-s is usually present among popcorn lines and annual teosintes whereas other allele forms (Ga1-m, ga1) are found in other maize germplasms (Kermicle et al.,
2006). Specifically, because of the dent sterility, crosses between dent corn and popcorn can only be made uni-directionally, using popcorn as the male parent.
4.5 Quality Protein Popcorn
For popcorn protein quality improvement, a crude attempt to increase popcorn grain lysine content was made by infusion popcorn with aqueous solutions of L-lysine and the lysine content was increased from 0.32% in unprocessed kernels to 3.0% in infused
(Blessin et al., 1971). Similar studies were conducted on fortification of wheat and dent corn (Blessin et al., 1970; Graham et al., 1968).
Breeding of Quality Protein Popcorn (QPP)
Several QPM conversions have been performed in dent corn (Babu et al., 2005;
Collard and Mackill, 2008; Manna et al., 2005; Surender et al., 2014).
Introgression of the o2 gene without modifier genes into popcorn backgrounds was carried out in several studies where popping was not maintained at all (Adunola,
2017; Zhou et al., 2016). To date, there have not been any reports of QPM conversion for popcorn lines that have maintained popping. In fact, introgression of any dent corn trait to popcorn was rarely reported. Dent corn by popcorn crosses were made mainly for mapping studies of quality trait QTLs for popcorn 56 improvement such as higher yielding hybrids and popping expansion volume
(PEV) (Dofing et al., 1991). The development of Quality Protein Popcorn (QPP) is challenging because it requires full QPM conversion but with the added challenges resulting from popcorn dent sterility and recovering acceptable popcorn quality. 57
Figure 1. Structure of a maize kernel (Hoseney et al., 1986).
58
Figure 2. Regulatory effects of the Opaque-2 gene (Prioul et al., 2008).
59
Figure 3. A proposed model for PBF and O2 controlling kernel nutritional quality and
yield in maize (Zhang et al., 2016). 60
Figure 4. Multiple pathways in which the 35 direct regulatory targets of O2 are involved
(Mach, 2015). 61
Figure 5. QTLs influencing free amino acid content w mapped using the F2 population
of Oh51Ao2 x Oh545o2 (Wang and Larkins, 2001). 62
Figure 6. The aspartate family biosynthetic pathway (Galili, 1995).
63
Figure 7. Enzymatic catabolism of lysine in seed (Galili, 1995).
64
Figure 8. Compatibility relationships among alleles of ga1 (Jones et al, 2015). 65 Table 1. Thirty-five genes downregulated in W22o2 compared with wild type with O2 binding site detected (Li et al, 2015). 66 Table 2. Some features of maize mutants affecting zein accumulation (Motto et al., 2003). 67 REFERENCES
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CHAPTER 2
DESIGN OF BREEDING SCHEME FOR QPP DEVELOPMENT
108 As consumers begin to pay attention to health and nutrition in their diet,
development of crop with increased nutrition is desirable. For the purpose of
increasing crop nutrition, transgenic approaches and conventional approaches have
been reported. Genetic transformation was applied to improve nutritional quality
in sorghum via introducing a mutated gene dhdps-r1 which codes for a feedback
insensitive dihydropicolinate synthase (Yohannes et al., 1999). Zein reduction by
transgenic means successfully increased total lysine, methionine, tryptophan and
aspartate and also digestibility in comparison to wild type (Fraley, 2009). In a
conventional non-transgenic approach to increase protein quality, QPM conversion
requires introgression of three distinct genetic systems, recessive mutant allele of
the o2 gene, the QTLs for endosperm vitreousness and for amino acid modifier
genes (Nedi et al., 2016). For introgression of o2, availability of O2 in-gene and
flanking markers can facilitate the introgression process of the o2 allele (Babu et
al., 2005; Babu and Prasanna, 2014; Kostadinovic et al., 2016; Krishna et al.,
2017).
The difficulties encountered in QPM conversion projects result from restoring kernel
vitreousness because of the complexity of modifier QTLs. Several mapping studies
were carried out and QTLs for modifiers (in terms of kernel vitreousness and density)
were mapped on Chromosomes 1, 5, 7 and 9 (Babu et al., 2015; Holding et al., 2011;
Holding and Larkins, 2008). Multiple lines of evidence suggest that an increase in gene expression and protein accumulation of 27-kD γ-zein plays a major role in modification in QPM and this gene resides within the major QTL on Chromosome 7
109 (Geetha et al., 1991; Holding, 2014; Holding and Larkins, 2008; Wu et al., 2010). The
characteristic increase has been used as a biochemical marker for endosperm
modification in QPM. Recently, it was verified that the 27-kD γ-zein protein increase
is conferred by a duplication event at 27-kD γ-zein locus (Liu et al., 2016). Causal
genes within other QTLs remain unknown. As a result, QPM conversion still relies
heavily on phenotypic selection of endosperm modification although the 27-kD γ-zein genetic and biochemical markers are useful. A previous QPM-conversion breeding program adopted both foreground (for o2) and background selection via MAS and developed QPM conversion lines within two backcrossing generations (Babu et al.,
2005; Kostadinovic et al., 2016). MAS has not been widely used in selection of
modifier genes because regions delineated as modifier QTLs tend to be large genetic
intervals and highly variable in effect. The most significant QTL, 27-kD γ-zein
duplication, is common across all QPMs and can now be used as a foreground marker
(Liu et al., 2016). Also, the fact that mapping studies of modifier QTLs had been limited to specific QPM germplasms might suggest that other minor effect QTLs are not common across all QPMs. This may restrict the application of MAS in projects such as this one in which multiple introgressions from different QPMs were
performed.
Development of Quality Protein Popcorn (QPP) has a number of major challenges.
Comparison between the B73 genome and the popcorn landrace Palomero Toluqueno revealed that Palomero genome is 22% smaller yet with larger predicted gene number
being around 58,000, compared with 50,000 for B73 (Schnable et al., 2009; Vielle-
Calzada et al., 2009; Walbot, 2008). Assessment of genetic diversity using inter-
110 simple sequence repeat (ISSR) showed that there is great amount of polymorphism
between dent and popcorn lines which are of different grouping after PCA and
cluster analysis (Kantety, 1995). Popcorn usually carries cross-incompatibility genes (known as gametophyte factors) that prevent popcorn from being fertilized by pollen of dent corns. This has been exploited for propagation of popcorn since it need not be grown in isolation from transgenic hybrid corn. Any popcorn breeding program must maintain this dent sterility. Additionally, breeding for popcorn improvement and trait introgression into popcorn germplasm requires maintenance of popping expansion volume (PEV). Studies mapping PEV in several
dent x popcorn populations indicate multiple and variable QTLs (Dhliwayo, 2008;
Li et al., 2007; Li et al., 2009) and show the complexity of PEV. Introgression of
dent traits into popcorn backgrounds is hindered by dent alleles with negative
effects on PEV.
In our study, we initiated the F1 cross using popcorn parents as males and QPM (dent
corn) as females because of dent sterility. A backcrossing strategy in which the popcorn
was the female was adopted to quickly recover the popcorn parent genome to ensure
recovery of PEV. Subsequent self-pollinations were performed to select for o2 mutants with acceptable modification. Materials, methods and the schematic breeding plan are provided as follows.
1. Materials and Methods
1.1 Plant Materials.
The 11 elite popcorn lines, whose identities are withheld, were provided by
111 ConAgra. For QPM lines, K0326Y, is a tropical QPM inbred line developed in
South Africa by Hans Gevers (Gevers and Lake, 1992). The other 11 QPMs were from North Central Regional Plant Introduction Station.
1.2 Methods
Total Zein Extraction
Zeins and non-zeins were extracted according to an established method (Wallace et al.,
1990). Briefly, 50 mg kernel flour was incubated overnight in 1ml of borate extraction
buffer (with 2% β-mercaptoethanol) with shaking at room temperature. After
centrifugation at room temperature for 15 min, supernatant was total protein extract.
Non-zein proteins were precipitated from 300 µl of total protein extract in 70% ethanol.
After centrifugation, the supernatant was the zein protein extract, which was dried in a
SpeedVac and resuspended in 200 µl of ddH2O. 5 µl was loaded for SDS-PAGE analysis.
DNA Isolation
Leaf tissue was collected from individuals in backcrossing generations (BC1 and
BC2) for DNA extraction. DNA extraction was carried out using a BioSprint 96
workstation from Qiagen according to the user manual.
Genotyping using O2 in-gene marker umc1066
PCR was carried out in 20µl volumes consisting of NEB Taq 0.15 µl, 0.2µM
dNTPs, 2µl standard Taq Reaction Buffer (10 x), 0.2µM forward/reverse primer of
umc1066, template DNA 50ng. PCR procedure was as follows: initial denaturation
112 at 94°C for 4 minutes followed by 35 cycles of three steps including 94°C for 30 seconds, 57 °C for 60s, 72°C for 60s. Final elongation was done at 72°C for 10 minutes. PCR products were visualized on 3-4% agarose gels. Individuals with O2o2 genotype were selected in backcrossing generations. This was also used for confirmation of o2 mutants in later generations (BC2F2, BC2F3, BC2F4).
Evaluation and selection of endosperm modification
Kernels were put embryo side down on a light box. Based on arbitrary visual estimation of the opaque endosperm proportion, kernels were classified into five types with Type I being fully vitreous, Type II being approximately 25% opaque,
Type III being approximately 50% opaque, Type IV being approximately 75% opaque and Type V being fully opaque (Vivek et al., 2008).
Amino acid profiling
Samples for amino acids profiling analysis were ground together into a fine flour.
Free amino acids (FAA) were extracted from 6-7 mg of the flour pooled from 3 kernels of 3 QPM lines, 4 popcorn lines and the six BC2F4 introgressions, while protein-bound amino acids (PBAA) were extracted from 3-4 mg. FAA were extracted and analyzed as described in detail in (Angelovici et al., 2013) using a
UPLC-MSMS system (Xevo TQ-S from Waters Corporation). For the analysis of protein-bound amino acids (PBAA), acid hydrolysis (Fountoulakis and Lahm,
1998) was performed prior to the FAA extraction and LC-MS/MS analysis described above. Briefly, 200 µl of 6N HCl was added to 4 mg of flour and incubated for 24 h at 110 °C. Ten micro liters (10 µl) were taken from the hydrolyzed samples
113 and dried using a Savant SpeedVac concentrator (Fisher Scientific) before
resuspension in the FAA extraction buffer and further analyzed as described
(Angelovici et al., 2013).
Quantitative measurements of popping volume
BC2F5 samples along with corresponding popcorn parents were prepared and
adjusted to same moisture content for quantitative popping. After popping
treatment in an Orville Redenbacher Hot Air Popcorn Popper, a 200 ml cylinder
was used to measure the total volume.
Total protein extraction and relative quantitation
Total protein extract (as described in Total Zein Extraction) from 50mg kernel flour
were diluted 250 times and 25 µl of the diluted solution were used for
Bicinchoninic Acid (BCA) protein assay. Protein concentration was assessed by the
BCA assay kit (Pierce) according to the user’s manual. Absorbances at (OD = 562 nm) were measured on a microplate reader Synergy2 (BioTek). Protein concentrations were determined from the BSA standard curve. Protein concentrations were compared between BC2F4 QPP introgressions with corresponding popcorn parents.
Statistical Analysis
Data were presented as Means ± SEM. P values less than 0.05 were considered significant. Principal Component Analysis (PCA) was carried out using R software.
2. Breeding Scheme and selection procedure 114 2.1 Breeding scheme
We initiated the introgression with two continuous backcrossing generations
followed by four selfing generations (Figure 1). Because of dent sterility, the F1
crosses were made uni-directionally with popcorn lines as the male parents. To maintain dent sterility, the resultant F1s were backcrossed onto respective popcorn
lines (recurrent parents). F2 populations were generated for evaluation of modifier transfer across different crosses. Individuals in backcrossing generations (BC1,
BC2) carrying the o2 allele were screened out for generation advancement to
BC2F2. BC2F2 kernels with partial modification were used for generation advancement to BC2F3. In the BC2F3 generation, kernels with increased
modification were selected and used for characterization of completely vitreous
popcorn-like o2 mutants. Candidate lines were advanced to BC2F4 generation.
Promising BC2F4 ears showing uniform endosperm modification were advanced to BC2F5.
2.2 Genotypic and phenotypic selection applied in the breeding process
Twelve accessions, labeled as ‘QPM’, were originally considered which were
CML154Q, CML157Q, CML158Q, BSBBo2, HQPSCB, HQPSSS, WIL500,
BSAAo2, Tx802, Tx807, Tx811, and K0326Y (Gevers and Lake, 1992). To confirm that these lines were in fact modified o2 mutants (low α-zein, high 27-kD γ-zein, and increased non-zein protein), we performed zein and non-zein profiling using
SDS-PAGE (Figure 2). We also measured other parameters including lysine content, time to flowering, and uniformity of endosperm vitreousness 115 (modification) (Table 1). Based on the above criteria, despite initially advancing
F1s for several QPMs, we ultimately proceeded with generation advancement for three ideal o2 donors, which were CML154Q, Tx807 and K0326Y. Though the characteristics seemed ideal for QPM HQPSCB, generation advancement was discontinued because of heterozygosity for the o2 allele within the original QPM seed (Figure 3). Zein profiles of the three chosen QPMs and 11 popcorn lines
(labeled as P1-P11 to preserve identity) are shown (Figure 4).
Genotyping was carried out for o2 polymorphism across popcorn, QPMs and F1
crosses (Figure 5). F2 populations were generated for evaluation of modifier transfer across different crosses (Table 2). A scale for endosperm opaqueness was
devised using a light box (Figure 6). From the p-value, the ratio of opaque to vitreous kernels in these F2 ears deviates significantly from 1:3 implying that some o2 mutant
kernels are fully or partially modified. F2 populations with significant p value were
promising for modifier transfer. Small roundish popcorn kernels were observed in F2 populations. Popcorn phenotype vitreous kernels were selected for both SDS-PAGE analysis and preliminary popping analysis to verify the presence of fully modified popcorn-like o2 mutants in the F2 population (Figure 7). A proportion of F2 popcorn-
like kernels that were fully vitreous were obviously modified o2 from their zein
profiles. Furthermore, when such popcorn vitreous kernels were heated, 100%
(including 25% o2/o2) popped showing proof-of-concept with only a single dose of popcorn. Individuals in backcrossing generations (BC1, BC2) carrying the o2 allele were screened by genotyping using umc1066 (Figure 5C). BC2F2 kernels harvested
were phenotyped on the light-box and were assigned a level of modification on a
116 scale defined in the F2 population (Figure 6). In terms of genotype, they can be o2 mutants with complete modification which is ideal. However they can also be
heterozygous (O2o2) or homozygous (O2O2) with wild type phenotype. In order to guarantee the presence of o2o2 genotype for later generation advancement without the need to genotype all kernels, we selected Type II and Type III semi-
opaque kernels from BC2F2 populations for SDS-PAGE zein profiling (Figure 8).
After Type II and Type III kernels were confirmed to be o2/o2 mutants, they were
advanced into BC2F3 populations. In the BC2F3 generation, ears with good modification were selected and used for characterization of completely vitreous
popcorn-like o2 mutants (Figure 9). Candidate lines were advanced to the BC2F4
generation.
117
Figure 1. Breeding scheme for quality protein popcorn. (A.Selection of Opaque-2
polymorphic markers between QPM and popcorn parental lines; Make F1 crosses uni-
directionally using popcorn as male parents. B. Confirm that umc1066 is a co-dominant
type of marker that can be used to differentiate O2O2, O2o2, and o2o2 genotypes;
Simultaneously self F1 and backcross it to popcorn parental line (recurrent parent) to get
F2 population and BC1 population. C. Assess modifier transfer using F2 which segregates
25% o2o2. Select vitreous popcorn-like kernels to identify o2 mutants by SDS-PAGE and determine whether they can pop at F2 stage. D. Identify BC1 individuals with O2o2 118 genotype in the BC1 generation; Carry out another generation advancement by
backcrossing O2o2 BC1 to popcorn parental lines to get BC2 population. E. Identify BC2 individuals with O2o2 genotype and advance them to BC2F2 population; Select type II and type III opaque kernels which are expected to be o2 mutants. Randomly select several such kernels to do both genotyping and SDS-PAGE zein profiling to confirm they are o2 mutants. Self-pollinate selected type II and type III opaque kernels to BC2F3 generation.
F. Select BC2F3 individuals with complete modification. Carry out genotyping and SDS-
PAGE on completely vitreous kernels to verify they are modified o2 mutants. For verified o2 mutants with complete modification, advance by self-pollination to BC2F4 generation.
G. Select ears by the following two criteria: 1. Ear characteristics similar to popcorn parents. 2. Uniform modification of kernels across the whole ear. Perform amino acid profiling and preliminary popping analysis on kernels from selected ears. H. Bulk up seeds for formal popping analysis.) 119
Figure 2. SDS/PAGE analysis of zein proteins (A) and non-zein proteins (B) in wild type, o2
mutant and 12 QPMs. (M: marker. 1. Wild type. 2. opaque-2 mutant. 3. QPM K0326Y. 4.
CML154Q. 5. CML157Q. 6. CML158Q. 7. BSBBo2. 8. HQPSCB. 9. HQPSSS. 10. WIL500.
11. BSAAo2. 12. Tx802. 13. Tx807 14. Tx811. 15. K0326Y). Compared with wild type (lane
1), all o2 mutants except for HQPSSS (lane 9) and WIL500 (lane 10) have α-zein reduction.
Compared with opaque-2 mutant (lane 2), all 12 QPMs have increased level of 27kD γ-zein.
Based on this analysis, two QPMs, WIL 500 and HQPSSS were not used as donor for o2 and modifiers for later introgression.
120
Figure 3. Genotyping results for six randomly selected seedlings of QPM HQPSCB. (M.
DNA marker. 1-6: six randomly selected individuals of HQPSCB.). Genotyping using marker umc1066 was carried out using DNA templates extracted from fresh leaf tissue of
HQPSCB seedlings from six randomly selected kernels. This results showed that ear for
HQPSCB was a mixture of kernels which were of homozygous genotype O2O2 (lane 1), homozygous genotype o2o2 (lanes 2, 4, 6) and heterozygous genotype O2o2 (lanes 3, 5) for opaque 2 locus.
121
Figure 4. Zein profile of three selected QPMs (CML154Q, K0326Y, Tx807) and 11 popcorn
lines (P1-P11). All QPMs have reduced amount of 22kD α-zein and increased amount of 27kD
γ-zein, consistent with the fact that they are modified o2. All popcorn kernels are wild type in
that they don’t have α-zein reduction. In terms of 27kD γ-zein, it seems that one popcorn line
(P11) may have increased amount of 27 kD γ-zein, which may facilitate endosperm modification. 123
A
B
C
Figure 5. Screening of five pairs of o2 markers (umc1066, phi112, bnlg1200, bnlg2160,
phi057) across 11 popcorn lines and six QPMs (A), umc1066 is a co-dominant marker (B) and
application of umc1066 in BC1 and BC2 generations for cross K0326Y x P3 (C). In A, in total, six QPMs were included because, F1 were successfully made using these six QPMs as female 123 parents (Table 1.). umc1066 was selected for foreground selection of opaque 2 because it gave
positive amplification for all lines, showed most polymorphism and is a co-dominant marker
indicating all three genotypes (O2O2, O2o2, o2o2). In B, the polymorphisms for F1 crosses
(K0326Y x P3, K0326Y x P5, CML154 Q x P2, CML154Q x P3, Tx807 x P2) with both
parents by the sides confirmed that umc1066 is a co-dominant marker which is useful to
differentiate genotypes (O2O2, O2o2, o2o2). In C, individuals in BC1 (up, lane 1-20) and BC2
(bottom, lane 21-40) between QPM K0326Y and P3 were genotyped using umc1066, heterozygous genotypes (O2o2) carrying the opaque 2 mutant allele were pointed out with red arrows. 124
Figure 6. Classification of endosperm vitreousness of F2 population (K0326Y x P2) where
Type I is fully vitreous and Type V is fully opaque. This classification facilitated evaluation of modifier transfer in F2 populations. It was also applied to BC2F2 populations for selection
of Type II and Type III opaque kernels which were confirmed to be o2 mutants. 125 A
B
Figure 7. Identification of completely modified poppable o2 mutants in F2 population from cross Tx807 x P7. In A, Q. Tx807, P, popcorn parent P7. 1-12 were kernels randomly selected from a pool of roundish fully vitreous kernels in F2 population from cross Tx807 x P7. Boxes and asterisks represent vitreous popcorn-like kernels, which were QPM since they have low
α-zein and high 27kD γ-zein. In B, fifteen kernels selected from the same pool of roundish fully vitreous kernels were used for preliminary popping. Although kernels used for popping are not the same kernels as in A tested for zein profile, these are by definition ~ 25% o2o2. 126
Figure 8. Confirmation of Type II and Type III opaque kernels in BC2F2 generation as o2 mutants with low α-zein, high γ-zein compared to corresponding popcorn parental lines in
Figure 4. Type II and Type III opaque kernels in BC2F2 generation were selected for generation
advancements. Three such kernels were randomly selected from each ear to test zein profile to
see if they are o2 and the seven crosses tested here are CML154Q x P3, CML154Q x P2,
K0326Y x P3, Tx807 x P2, K0326Y x P3, K0326Y x P5, K0326Y x P10. From the zein protein profile, these kernels were confirmed to be o2 mutants because of reduction of 22kDα-zein,
they have increased 27 kD γ-zein which is consistent with the fact that these kernels are modified.
127 A
B
C.
Figure 9. Presence of Type I (vitreous) o2 mutants in BC2F3 populations from partially modified (Type II and Type III opaqueness) BC2F2 individuals. A. Self-pollinating partially
128 modified o2 mutants selected from BC2F2 generation can give rise to BC2F3 population with increased modification. Examples from six introgressions which were CML154Q x P3,
CML154Q x P2, CML154Q x P9, Tx807 x P2, K0326Y x P7, K0326Y x P3 were included here. For each of these introgressions, 20 Type II and Type III opaque BC2F2 kernels selected for planting in 2016 summer were shown on the left. After self-pollination, light box phenotyping was carried out on resultant BC2F3 populations. To compare endosperm modification with Type II and Type III BC2F2 individuals, best modified BC2F3 ear for each cross was selected and fully vitreous kernels from this ear were shown on the right (right, in circles) for each introgression. Same selection was applied to introgression from F1 cross
K0326Y x P5, which were not included in this figure because of only 10 kernels in BC2F2 population were selected as Type II and Type III opaque kernels. B. Confirmation of vitreous
BC2F3 individuals to be o2 mutants. For cross CML154Q x P3, five promising ears were selected in BC2F3 generation. To confirm that these kernels with increased modification were o2 mutants, three such kernels were randomly selected from each ear. The results showed that all these kernels are o2 mutant. C. Confirmation of vitreous kernels in the BC2F3 generation to be o2 mutants with reduced α-zein and increased 27kD γ-zein to guarantee modification. Three kernels with Type I opaqueness were selected from each promising
BC2F3 population. There seemed to be variation between individuals in terms of 27kD γ-zein expression.
129 Table 1. Characteristics of the 12 QPMs originally considered. Vitreous/ Opaque Kernel F1 Relative time to pedigree Source Zein content Lysine color made flowering Mostly vitreous, CML154Q Ames27083 white Yes 2 weeks late High γ, Low α 0.37±0.06 smaller, roundish 3 weeks late, no CML157Q Ames27084 Mostly vitreous white No High γ, Low α NA pollination 4 weeks late, no CML158Q Ames27085 All fully vitreous white No High γ, Low α NA pollination Mix of vitreous, BsBBo2(S)C2 PI550495 yellow Yes Normal High γ, Low α 0.45±0.06 semi-vitreous,
HQPSCB PI586689 Fully vitreous, large yellow Yes 1-2 weeks late High γ, Low α 0.35±0.06
HQPSSS PI586690 Fully vitreous, large yellow Yes Normal High γ, High α 0.34±0.04 Vitreous, large, WIL500 PI601689 yellow Yes 2 weeks late High γ, High α 0.36±0.02 roundish BSAAo2(S)C1 PI608781 Mostly opaque yellow Yes Normal High γ, Low α 0.44±0.06 All modified, smaller Tx802 PI619429 yellow No 2 weeks late High γ, Low α 0.42±0.02 kernel All super vitreous, Tx807 PI619430 white Yes 2 weeks late High γ, Low α 0.45±0.01 smaller kernel Mix of vitreous and 3 weeks late, no Tx811 PI619431 white No High γ, Low α NA semi-vitreous pollination A tropical 3 weeks late, no K0326Y QPM inbred All vitreous, roundish yellow Yes High γ, Low α 0.39±0.01 developed pollination b H
Note. Highlighted in yellow are the QPM parents for ongoing QPP introgressions. Amino
acid quantification was carried out by Agricultural Experiment Station Chemical
Laboratories in University of Missouri-Columbia in 2014. For lysine measurement, NA =
‘not measured’ (for CML157Q, CML158Q, Tx811). For these three QPM lines, no cross was
made because of difference in time to flowering. Lysine measurements for comparison:
lysine measurement for unmodified o2 mutant B73o2 is 0.60±0.02. Lysine measurement for
B73 wildtype is 0.32±0.01.
130
Table 2. Evaluation of modifier transfer at F2 stage Number of observed Number of opaque kernels observed vitreous Chi-square test χ2 Germplasm (Type III ~ Type V) kernels (df=1) p-value CML154 x P2 178 1695 239.89 2.20E-16* CML154Q x P3 147 1069 108.11 2.20E-16* CML154Q x P9 231 1471 118.54 2.20E-16* K0326Y x P6 273 854 0.36232 0.5472 K0326Y x P4 227 714 0.38576 0.5345 K0326Y x P10 308 1058 4.3816 0.03633* K0326Y x P7 181 872 34.264 4.81E-09* K0326Y x P5 277 922 2.3022 0.1292 K0326Y x P3 188 806 19.639 9.35E-06* Tx807 x P2 281 1050 10.731 0.001054* Tx807 x P7 220 902 17.399 3.03E-05*
Note. Three F2 ears were randomly selected for each cross. Number of opaque kernels (Type
III ~Type V) and number of vitreous kernels were summed up respectively from three ears.
Theoretically, it there is no modifier, opaque2 would be segregating 1:2:1 (o2/o2 : O2/o2 :
O2/O2) genotypically resulting phenotypically segregating for endosperm modification with a ratio of 1:3 (opaque : vitreous). Deviation of the ratio for endosperm modification from 1:3 reflects the effect of modifier in the F2 stage. Chi-Square analysis was carried out for pools of
three F2 populations for 11 crosses. The result was summarized in Table 2. Significant p– values (α=0.05) was indicated by asterisk (*). Crosses with advanced BC2F4 introgressions were highlighted in bold. For cross K0326Y x P10, only five BC2F2 ears were harvested to
select from and none gave enough kernels with Type II and Type III modification. For cross
K0326 Y x P7, one promising BC2F2 ear was selected out of 14 ears but selection stopped at
BC2F3 stage because there were no signs for improved modification. For cross Tx807 x P7, six
ears were harvested and none of them showing acceptable modification. For four of the six
QPP introgressions in our study which were from cross CML154Q x P2, CML154Q x P3,
K0326Yx P3 and CML154Q x P9, at least four BC2F2 ears segregating for endosperm
131 modification were received for selection of Type II and Type III kernels from. Three such
BC2F2 ears were selected for QPP6 (from cross Tx807 x P2). Two such BC2F2 ears were selected for QPP5 (from cross K0326Y x P5). 132 REFERENCES
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CHAPTER 3
ANALYSIS OF QPP DEVELOPED BETWEEN THREE QPM AND FOUR
POPCORN PARENTAL LINES
137 1. Selection of BC2F4 population with full modification
From 2017 summer harvest, we selected ears in BC2F4 population with popcorn
appearance and uniform modification across the cob (Figure 1). In total six BC2F4 introgressions between three QPM parents and four popcorn parents were selected, which are QPP1, introgression from cross K0326Y x P3; QPP2, introgression from cross CML154Q x P2; QPP3, introgression from cross CML154Q x P9; QPP4,
introgression from cross K0326Y x P3; QPP5, introgression from cross K0326Y x P5;
QPP6, introgression from cross Tx807 x P2, respectively.
2. Confirmation of o2 genotype in BC2F4 population with full modification
SDS-PAGE analyses (Figure 2) and umc1066 genotyping results of BC2F3 (Figure 9 in
Chapter 2) confirmed that all kernels were modified o2 mutants. Consistent with their
vitreous appearance, they all have increased 27-kD γ-zein protein with respect to the wild
type (popcorn parent) control. The uniformity in enhanced expression of 27-kD γ-zein
implies that phenotypic selection alone is sufficient for the retention of this modifier QTL.
BC2F4 ears from selected modified Type I o2 kernels show more uniform modification than
the BC2F3 population, where there was more variability for modification in the ears.
Because of lack of kernels for the Tx807 introgression which is also at an intermediate
stage of modification restoration (Figure 3), it is not included for SDS-PAGE confirmation
here (but included in later amino acids profiling to see the lysine difference).
3. Amino acids profiling revealed high lysine in all six BC2F4 o2 introgressions
In order to determine whether these popcorn-like o2 mutants have the high quality protein
138 phenotype, amino acid profiling (both protein-bound amino acid and free amino acid) was carried out for six introgressions and parental lines including three QPM parents and four popcorn parents (Angelovici et al., 2013; Fountoulakis and Lahm, 1998). We also included
B73 wild type and B73o2 as dent corn controls for comparison to modified BC2F4 samples.
Vitreous endosperm content for the profiled kernels was shown by both light transmittance and by cutting kernels transversely (Figure 3). Here we included Tx807 BC2F4 individuals, whose endosperm modification was not as advanced as other introgressions. Compared with wild type popcorn parental lines (P3, P2, P5, P9), all o2 mutants (QPMs, QPP introgressions) have a small opaque center. Compared with QPP1-QPP5, the amount of the central opaque region of QPP6 is larger. This is consistent with the fact that it is only partially modified currently and requires selection in future generation advancements.
QPP1 and QPP4 had the least central opaque region, consistent with their vitreous appearance. For QPP3 and QPP4, the kernels had a slightly altered distribution of the opaque region. In total, sixteen amino acids were detectable and quantified for each sample.
The nine kernels shown for each genotype represent three pools of three kernels (three biological replicates). The sixteen amino acids measurable were Ala, Arg, Aspx, Glx, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr and Val (Table 1) where Glx is both glutamic acid and glutamine.
The results showed that all six introgressions have significantly increased content of both free lysine and protein-bound lysine compared with corresponding popcorn parents (Figure
4AB). The ratio of protein-bound lysine between QPPs and corresponding popcorn parental lines ranges from 1.45 fold (QPP5 compared with P5) to two-fold (QPP4 compared with P3) (Table 1A). For free lysine, the ratio ranges from 4.05 fold (QPP3
139 compared with P9) to 12.3 fold (QPP6 compared with P2) (Table 1B). To extrapolate and display the global variation for all amino acids measured, we adopted the Principal
Component Analysis (PCA) used in multivariate analysis (Figure 4C, Figure 5). PCA using protein-bound amino acids data showed that the six introgressions grouped together with
QPM germplasms in a distant group from popcorn parental lines (Figure 4C). This result suggests that the six introgressions have amino acid profiles similar to QPM. From the
PCA biplot, the 15 amino acid (15 variables indicated as vectors) have different coordinations, implying different contribution to the total variation. Among the ones pointing toward the o2 group, four amino acids (Lys, His, Aspx, Arg) were positively correlated with o2 lines, whereas Leu, Tyr, Ile and Ala were positively correlated with wild type germplasms. PCA analysis of free amino acids was also carried out and no clear pattern was observed across different o2 mutants (QPM parents, QPPs and B73o2) (Figure
5). Previously, whole kernel amino acids contents were compared between o2 and wild type maize and showed the general lysine increase resulting from globally increased non- zein proteins (Hunter et al., 2002). Here, we compared the ratio of o2 by wild type for 15 amino acids measured both in the above study and this study to see whether the above eight amino acids showed similar pattern between all o2 and wild type samples (Figure 6). This showed that the log2 value of the ratio of amino acid (o2 by wild type) is consistently positive for Lys, His and Arg, suggesting there is more of these amino acids in o2 background than in wild type backgrounds. In contrast, the log2 value of the ratio of amino acid (o2 by wild type) was consistently negative for Met, Ser, Glx, Ala, Ile, Leu and Phe.
In both PCA biplot and comparative analysis of protein bound amino acid profile, Leu, Ile,
Ala stand out as amino acids that are reduced in the o2 background compared with wild
140 type. Results of BCA protein assay on BC2F4 populations indicated that there was no reduction in total protein in all six QPP germplasms (Figure 7). Specifically, for QPP3 and
QPP4, significant increases of total protein (α=0.05) was observed compared with corresponding popcorn parental lines.
4. Selection of fully modified o2 popcorn can result in equivalent popping to popcorn parents
Small scale qualitative popping analysis on BC2F4 kernels confirmed them to be fully poppable (not shown). After further bulking, BC2F5 kernels were used for small scale quantitative popping analysis (Figure 8A). To represent a general popping ability, three ears were selected which were the progeny of the BC2F4 ears used for amino acid profiling.
Three pools of kernels (3.33g each) were used as biological replicates for each ear.
Percentages of popped kernels were above 90% for all QPP and Popcorn parental lines
(Table 2). Volume of the popped kernels were measured and used for pair-wise comparison with corresponding popcorn parental line (Table 2, Figure 8B). Comparisons that exhibited no significant difference are highlighted in bold in Table 2 and bracketed in Figure 8B.
There were no significant differences in the popping volume scores for QPP1-2 vs P3 comparison (t(3.2) = 0.50, p=0.6495), QPP4-1 vs P3 comparison (t(3.93) = -2.71, p =
0.0544) and QPP2-3 vs P2 comparison (t(3.92) = -2.47, p = 0.0699). Popping volumes equivalent to corresponding popcorn parents were achieved in these BC2F5 populations
(QPP1-2, QPP4-1, QPP2-3). For QPP1 and QPP4, where comparable popping volume was observed, the least residual proportion of opaque endosperm was observed. For QPP6, a mid-way introgression for which future selection of endosperm modification is required,
141 the large residual region of opaque endosperm is contributing to reduced popping volume.
142
Figure 1. Kernel and zein phenotype of BC2F4 populations. A. Promising BC2F4 introgressions
143 with uniform modification. In total, two ears were selected for each of the five introgressions,
which were originated from crosses CML154Q x P3, CML154Q x P2, K0326Y x P3, K0326Y
x P5 and CML154Q x P9 respectively. Tx807 introgression (originated from Tx807 x P2) was
not included because the ear was not fully filled. Compared with the other five introgressions
in kernel modification, this cross was a little behind in that the kernels were between Type I
and Type II opaqueness (Fig.3). For Tx807 introgression, BC2F4 individuals were prepared for amino acid profiling analysis, the rest were self-pollinated to restore modification. BC2F5 populations for this cross were included in quantitative popping analysis. For each of the five crosses, two independent introgressions were carried onward with. The ones marked with asterisks were selected for amino acid profiling analysis.
144
Figure 2. SDS-PAGE confirmation of o2 mutants in vitreous BC2F4 populations.
Confirmation of completely modified o2 mutants for introgressions from cross K0326Y x P3
(upleft), cross CML154Q x P9 (upright), cross K0326Y x P3, cross CML154Q x P2 (bottom left), CML154Q x P3 (bottom middle) and K0326Y x P5 (bottom right) are shown here.
145
Figure 3. QPM, popcorn and six BC2F4 introgressions prepared for amino acid profiling. Left, kernel modification on Light box. Right, kernel dissection. QPP1, introgression from cross K0326Y x P3; QPP2, introgression from cross CML154Q x P2; QPP3, introgression from cross CML154Q x P9; QPP4, introgression from cross K0326Y x P3; QPP5, introgression from cross K0326Y x P5;
QPP6, introgression from cross Tx807 x P2.
146
Figure 4. Confirmation of high protein quality of six QPP BC2F4 introgressions in comparison to
QPM (CML154Q, K0326Y, and Tx807) and popcorn (P3, P2, P5 and P9) controls. A. free lysine
comparison. B. protein-bound lysine comparison. C. PCA analysis of protein bound amino acids.
Principal component scores (PC1 and PC2) from each observation were plotted as dots in different
shapes. Amino acids (variables) were plotted as arrows. 147
Figure 5. PCA-biplot for QPM, popcorn and six BC2F4 introgressions using free amino acid
measurements. PCA of free amino acid data showed that all wild type germplasms grouped together. Three QPMs were distant with the wild type group. This shows variation between three QPMs and no obvious pattern for o2 germplasms in terms of free amino acids.
148
Figure 6. Comparison of log2 value of ratio (o2 / wildtype ratio) for 15 amino acids using protein- bound amino acid measurements. Horizontal axis showed the 15 amino acids shared commonly between our protein bound amino acid data and Hunter’s data which are respectively Asx (both
149 aspartic acid and asparagine), Met, Thr, Ser, Glx (both glutamic acid and glutamine), Pro, Gly,
Ala, Val, Ile, Leu, Phe, His, Lys and Arg. The 14 bars in each of the fifteen amino acids
represent the log 2 value of the ratio for the amount of certain amino acid between o2 and wild type in Hunter’s data, B73o2 and B73 wild type, QPP1 (introgression from cross CML154Q x P3) and P3, CML154Q and P3, QPP2 (introgression from cross CML154Q x P2) and P2,
CML154Q and P2, QPP3 (introgression from cross CML154Q x P9), CML154Q by P9, QPP4
(introgression from cross K0326Y x P3) and P3, K0326Y x P3, QPP5 (introgression from cross K0326Y x P5) and P5, K0326Y x P5, QPP6 (introgression from cross Tx807 x P2) and
P2, Tx807 x P2. Amino acids (Met, Leu, Phe, His, Lys, Arg) with uniform pattern (with log2 of the ratio being all positive or negative) across 14 comparisons implies their contribution to o2 and wild type difference in general and lysine is one of them. The fact that 11 out of 15 amino acids showed uniform pattern across all 14 comparisons implies the change of proteins is similar across o2 and wild type germplasms.
150
Figure 7. Comparison of relative concentration for total protein between BC2F4 QPP introgressions
and corresponding popcorn parental lines. Average of total protein in six QPPs were considered as 1. Relatively, the amount of total protein in corresponding popcorn parental lines were indicated as ratio to corresponding QPPs. For QPP1, the increase in total protein is significant when compared with corresponding popcorn parent P3 (p=0.03974). Significant increase was found in QPP4 compared with corresponding popcorn parent P3 (p=0.0166) (α=0.05). 151
Figure 8. Comparison of popping characteristics between QPP introgressions with corresponding
popcorn parents. A. kernels before and after popping (QPP1, BC2F4 introgression from cross
CML154Q x P3; QPP4, BC2F4 introgression from cross K0326Y x P3; QPP2, BC2F4 introgression from cross CML154Q x P2. B. Popping volume comparison between QPPs and corresponding popcorn parental lines. Brackets between a popcorn parent and a QPP progeny indicate no significant differences.
15
Table 1. Amino acid profile of QPMs, popcorn parents, and six different introgressions (BC2F4). A. Protein-bound amino acids.
AAa CML154Q K0326Y Tx807 P3 P2 P5 P9 QPP1 QPP2 QPP3 QPP4 QPP5 QPP6 B73 wt B73 o2 Ala 0.55±0.03 0.51±0.01 0.60±0.04 0.67±0.02 0.85±0.10 1.04±0.10 0.89±0.05 0.52±0.07 0.65±0.01 0.48±0.02 0.53±0.02 0.61±0.06 0.72±0.05 0.96±0.04 0.80±0.11 Arg 0.29±0.04 0.25±0.02 0.34±0.02 0.18±0.01 0.21±0.01 0.21±0.01 0.20±0.02 0.25±0.00 0.30±0.01 0.25±0.00 0.29±0.01 0.29±0.01 0.32±0.01 0.45±0.05 0.76±0.08 Asx 0.79±0.02 0.72±0.04 1.04±0.09 0.58±0.03 0.72±0.01 0.78±0.01 0.71±0.07 0.89±0.03 0.95±0.04 0.70±0.03 0.70±0.02 0.99±0.07 1.18±0.03 0.69±0.04 1.18±0.07 Glx 1.40±0.38 1.10±0.07 1.40±0.09 1.46±0.05 1.67±0.08 1.80±0.03 1.72±0.20 1.26±005 1.40±0.06 1.17±0.00 1.23±0.04 1.42±0.04 1.45±0.01 2.21±0.11 1.76±0.25 Gly 0.57±0.11 0.46±0.01 0.69±0.14 0.74±0.53 0.47±0.11 0.51±0.09 0.63±0.16 0.43±0.08 0.50±0.13 0.66±0.17 0.47±0.08 0.60±0.17 0.63±0.25 0.84±0.08 0.85±0.03 His 0.31±0.01 0.31±0.01 0.37±0.01 0.20±0.01 0.25±0.01 0.26±0.01 0.27±0.03 0.32±0.02 0.40±0.01 0.35±0.01 0.38±0.01 0.36±0.01 0.40±0.02 0.27±0.01 0.34±0.03 Ile 0.39±0.01 0.38±0.01 0.50±0.02 0.51±0.02 0.63±0.03 0.67±0.00 0.62±0.06 0.42±0.03 0.47±0.01 0.41±0.01 0.45±0.02 0.50±0.02 0.57±0.02 0.48±0.03 0.44±0.07 Leu 0.89±0.06 0.83±0.03 1.06±0.08 1.50±0.03 1.74±0.06 1.97±0.01 1.80±0.22 0.95±0.10 1.05±0.08 0.88±0.04 1.01±0.04 1.20±0.02 1.25±0.06 1.50±0.08 0.98±0.16 Lys 0.31±0.01 0.29±0.02 0.41±0.04 0.16±0.02 0.24±0.01 0.22±0.01 0.19±0.02 0.29±0.00 0.38±0.02 0.30±0.01 0.32±0.01 0.32±0.02 0.43±0.01 0.24±0.02 0.48±0.04 Met 0.07±0.02 0.06±0.01 0.07±0.01 0.09±0.02 0.10±0.00 0.07±0.02 0.09±0.00 0.04±0.00 0.06±0.01 0.04±0.01 0.04±0.00 0.05±0.00 0.04±0.01 0.26±0.01 0.19±0.02 Phe 0.36±0.02 0.32±0.01 0.44±0.02 0.52±0.03 0.61±0.05 0.70±0.02 0.63±0.08 0.36±0.03 0.40±0.02 0.33±0.01 0.37±0.02 0.43±0.02 0.50±0.03 0.56±0.05 0.50±0.11 Pro 0.83±0.04 0.82±0.02 0.96±0.02 0.89±0.05 1.03±0.03 1.08±0.04 1.05±0.11 0.89±0.06 1.06±0.06 1.04±0.06 0.98±0.04 1.03±0.05 1.12±0.03 0.85±0.04 0.69±0.07 Ser 0.40±0.02 0.40±0.01 0.51±0.02 0.47±0.03 0.57±0.00 0.61±0.01 0.57±0.07 0.42±0.02 0.48±0.01 0.39±0.01 0.46±0.01 0.48±0.02 0.52±0.03 0.56±0.03 0.55±0.06 Thr 0.32±0.02 0.32±0.03 0.42±0.03 0.33±0.02 0.45±0.05 0.45±0.03 0.43±0.07 0.37±0.03 0.44±0.02 0.36±0.03 0.41±0.02 0.42±0.02 0.53±0.03 0.54±0.02 0.52±0.06 Tyr 0.18±0.06 0.18±0.01 0.24±0.02 0.27±0.00 0.32±0.00 0.33±0.01 0.31±0.04 0.19±0.01 0.23±0.02 0.19±0.01 0.20±0.01 0.23±0.00 0.25±0.01 0.27±0.01 0.21±0.02 Val 0.51±0.01 0.50±0.02 0.62±0.01 0.46±0.02 0.59±0.02 0.62±0.01 0.57±0.06 0.53±0.04 0.63±0.02 0.54±0.00 0.58±0.00 0.59±0.02 0.65±0.02 0.54±0.02 0.64±0.06
Note: AAa, protein bound amino acids of sixteen amino acids (g/100g flour). The acid hydrolysis method used lead to the destruction of tryptophan which is therefore not detectable. Amino acids content refers to mean ± standard deviation (n=3). B73 wt and B73 o2 were included for comparison. Table 1. Continued.
B. Free amino acids 15
b AA CML154Q K0326Y Tx807 P3 P2 P5 P9 QPP1 QPP2 QPP3 QPP4 QPP5 QPP6 B73 wt B73 o2 Ala 1.44E-02± 7.29E-03± 3.60E-02± 2.21E-03± 3.10E-03± 1.15E-02± 6.77E-03± 2.38E-02± 4.35E-02± 6.69E-03± 5.37E-03± 4.49E-02± 1.85E-02± 7.71E-03± 2.49E-02± 1.47E-03 7.31E-04 7.47E-03 1.45E-04 3.61E-04 1.88E-03 2.31E-03 4.79E-03 2.43E-02 3.73E-04 1.02E-03 5.89E-02 2.52E-03 3.99E-03 5.30E-03 Arg 4.23E-02± 1.63E-02± 1.49E-02± 2.82E-03± 3.76E-03± 2.14E-03± 1.75E-03± 1.52E-02± 2.16E-02± 1.13E-02± 1.16E-02± 3.18E-02± 3.76E-02± 5.00E-03± 1.63E-02± 3.43E-03 1.78E-03 3.10E-03 5.20E-04 3.79E-04 8.09E-05 4.42E-04 2.58E-03 5.17E-03 1.16E-03 1.67E-03 6.04E-02 3.80E-03 1.98E-03 7.20E-03 Asn 1.54E-01± 1.01E-01± 1.67E-01± 1.79E-02± 1.99E-02± 1.78E-02± 3.43E-02± 2.03E-01± 1.15E-01± 9.01E-02± 7.80E02± 2.25E-01± 2.52E-01± 1.45E-02± 4.88E-02± 1.86E-02 7.63E-03 2.78E-02 2.17E-03 3.81E-03 3.92E-03 1.29E-02 2.87E-02 9.58E-03 9.91E-03 1.30E-02 6.21E-02 1.56E-02 5.66E-03 1.87E-03 Asp 7.59E-02± 7.45E-02± 9.10E-02± 4.89E-03± 5.95E-03± 1.05E-02± 1.31E-02± 1.47E-01± 1.55E-01± 7.06E-02± 2.94E-02± 1.05E-01± 1.25E-01± 1.05E-02± 1.66E-01± 7.46E-03 8.42E-03 7.11E-03 7.36E-04 3.39E-03 9.86E-04 4.14E-03 1.41E-02 3.93E-03 9.70E-04 5.01E-03 3.96E-02 7.05E-03 3.53E-03 3.24E-02 Gln 3.83E-03± 7.43E-04± 1.52E-02± 1.04E-03± 1.05E-03± 3.27E-03± 3.84E-03± 5.88E-02± 4.77E-02± 1.83E-02± 1.50E-03± 2.79E-02± 3.29E-02± 2.20E-03± 6.83E-03± 2.79E-03 7.46E-05 8.85E-03 2.89E-04 3.44E-04 5.42E-04 1.79E-03 1.94E-02 2.71E-02 4.39E-03 2.06E-04 3.49E-02 1.68E-02 1.19E-03 4.14E-04 Glu 2.89E-02± 2.07E-02± 9.39E-02± 9.03E-03± 1.41E-02± 9.52E-03± 2.03E-02± 1.55E-01± 1.33E-01± 8.04E-02± 1.85E-02± 1.18E-01± 1.04E-01± 1.94E-02± 5.32E-02± 1.17E-02 2.37E-03 3.58E-02 1.43E-03 5.22E-04 1.29E-03 4.77E-03 8.66E-03 4.11E-02 1.38E-02 1.68E-03 3.59E-02 2.28E-02 9.54E-03 5.20E-03 His 6.33E-03± 5.69E-03± 6.30E-03± 1.06E-03± 1.68E-03± 1.72E-03± 1.20E-03± 4.14E-03± 7.18E-03± 3.07E-03± 3.54E-03± 9.65E-03± 9.45E-03± 1.28E-03± 5.79E-03± 8.04E-04 3.19E-04 8.54E-04 1.94E-04 1.46E-05 6.18E-05 9.28E-05 2.81E-04 1.50E-03 1.83E-04 3.83E-04 2.33E-02 4.49E-04 3.50E-04 1.31E-03 Ile 5.54E-04± 8.10E-04± 2.61E-03± 2.37E-04± 2.63E-04± 5.50E-04± 7.08E-04± 1.70E-03± 1.29E-03± 5.93E-04± 3.00E-04± 2.14E-03± 4.91E-04± 3.77E-04± 1.03E-03 1.50E-04 6.38E-05 2.99E-04 8.74E-05 9.18E-05 5.45E-05 8.04E-05 5.04E-04 5.67E-04 1.73E-04 7.90E-06 2.43E-02 8.92E-05 7.01E-05 ±1.05E-04 Leu 9.21E-04± 9.53E-04± 2.67E-03± 2.83E-04± 3.15E-04± 5.97E-04± 7.08E-04± 3.50E-03± 3.39E-03± 7.85E-04± 3.33E-04± 2.49E-03± 1.17E-03± 4.26E-04± 9.26E-04± 2.14E-04 6.08E-05 1.23E-04 9.51E-06 2.74E-06 1.04E-04 8.04E-05 8.23E-04 1.87E-03 8.35E-05 5.86E-05 2.52E-02 6.73E-04 1.55E-05 1.54E-04 Lys 1.53E-02± 9.39E-03± 1.39E-02± 1.47E-03± 2.28E-03± 1.56E-03± 1.24E-03± 6.38E-03± 1.07E-02± 5.03E-03± 6.19E-03± 1.04E-02± 2.81E-02± 1.52E-03± 8.51E-03± 2.19E-03 8.66E-04 1.96E-03 2.77E-04 3.69E-04 1.04E-04 6.04E-05 7.70E-04 3.56E-03 6.31E-04 7.24E-04 1.77E-03 2.56E-03 1.99E-04 3.43E-03 Met 8.37E-04± 1.63E-04± 1.39E-03± 1.61E-04± 1.0E-04± 3.44E-04± 4.04E-04± 2.20E-03± 9.08E-04± 3.35E-04± 2.14E-04± 2.26E-03± 3.33E-04± 2.67E-04± 7.63E-04± 2.34E-04 5.56E-06 3.43E-04 5.41E-06 0.00E-00 2.75E-05 1.07E-04 7.11E-04 6.91E-04 1.46E-05 7.50E-05 2.78E-02 1.65E-04 8.55E-05 1.23E-04 Phe 4.40E-03± 1.14E-03± 3.00E-03± 4.13E-04± 7.93E-04± 6.93E-04± 8.29E-04± 3.93E-03± 4.68E-03± 2.60E-03± 3.85E-04± 3.31E-03± 2.97E-03± 5.94E-04± 1.75E-03± 1.03E-03 1.82E-04 3.87E-05 8.83E-05 6.90E-06 6.86E-05 1.25E-04 4.65E-04 8.38E-04 2.98E-04 1.66E-05 2.92E-02 8.38E-04 8.90E-05 4.95E-04 Pro 1.52E-01± 8.43E-02± 1.43E-01± 8.98E-03± 1.02E-02± 2.53E-02± 3.55E-02± 1.20E-01± 1.00E-01± 1.53E-01± 2.44E-02± 8.13E-02± 2.07E-02± 4.54E-02± 6.47E-02± 1.34E-02 7.79E-03 1.59E-02 2.56E-03 1.10E-03 4.42E-03 1.80E-02 1.00E-02 4.06E-03 4.94E-03 5.05E-03 3.08E-02 1.26E-02 3.31E-02 2.28E-02 Ser 2.77E-03± 5.84E-03± 8.29E-03± 2.15E-03± 3.45E-03± 4.47E-03± 3.65E-03± 1.16E-02± 9.82E-03± 3.43E-03± 1.39E-03± 1.18E-02± 6.40E-03± 6.41E-04± 2.80E-03± 6.39E-05 5.66E-04 2.09E-03 2.04E-04 3.46E-04 7.93E-04 5.56E-04 4.44E-03 5.64E-03 3.84E-04 4.41E-04 7.03E-03 1.43E-03 1.57E-04 6.79E-04 Trp 3.65E-03± 1.56E-03± 1.58E-03± 6.60E-04± 6.54E-04± 8.57E-04± 7.09E-04± 2.35E-03± 2.71E-03± 2.06E-03± 1.40E-03± 2.52E-03± 2.21E-03± 5.81E-04± 2.00E-03± 4.93E-04 5.32E-05 1.60E-04 2.22E-05 1.44E-04 8.48E-05 2.04E-05 1.02E-04 1.33E-04 8.96E-05 3.69E-05 7.42E-03 7.59E-05 3.21E-04 3.30E-04 Thr 3.84E-03± 3.16E-03± 9.98E-03± 1.07E-03± 1.95E-03± 2.44E-03± 4.77E-03± 1.13E-02± 7.91E-03± 3.44E-03± 1.86E-03± 1.66E-02± 6.60E-03± 4.29E-04± 1.69E-03± 6.40E-04 2.03E-04 2.58E-03 1.79E-04 4.03E-04 4.33E-04 7.32E-04 1.58E-03 3.53E-03 8.32E-04 2.55E-04 7.70E-03 1.96E-03 6.99E-05 5.38E-04 Tyr 1.69E-02± 2.96E-03± 4.63E-03± 7.81E-04± 1.09E-03± 1.24E-03± 1.12E-03± 4.89E-03± 6.15E-03± 7.88E-03± 1.29E-03± 9.76E-03± 4.28E-03± 1.94E-03± 9.23E-03± 3.63E-03 9.77E-05 8.13E-04 2.63E-05 9.46E-06 1.10E-04 1.41E-04 2.86E-04 8.83E-04 6.40E-04 9.76E-05 5.78E-03 5.39E-04 6.47E-04 2.08E-03 Val 2.73E-03± 2.21E-03± 8.12E-03± 5.69E-04± 7.93E-04± 1.46E-03± 1.93E-03± 6.43E-03± 5.95E-03± 1.88E-03± 1.19E-03± 7.32E-03± 3.41E-03± 1.27E-03± 4.31E-03± 1.82E-05 2.35E-04 1.18E-03 1.92E-05 6.90E-06 2.65E-04 4.61E-04 9.90E-04 2.72E-03 1.71E-04 1.09E-04 5.76E-03 8.04E-04 5.92E-04 7.30E-04
Note: AAb, free amino acids (g/100g flour). Amino acids content refers to mean ± standard deviation (n=3). Gly and Cys measurements were not included because of missing values or reading below detection level. B73 wt and B73 o2 were included for comparison.
154 Table 2. Popping volume (ml) comparison and percentages of popped kernels between BC2F5 populations and popcorn parental lines.
t-test for Equality of Means Percentages of popped kernels
Mean Std. Std. 95% Confidence t df Sig. Difference Deviation Error Interval of the (2-tailed) Difference Difference Difference Lower Upper
QPP1-1 vs P3 -18.33 5.77 3.33 -28.58 -8.09 -5.50 3.20 0.0099 QPP1-2 vs P3 1.67 5.77 3.33 -8.58 11.91 0.50 3.20 0.6495 96.14% vs 100% QPP1-3 vs P3 -12.67 6.67 3.85 -25.26 -0.07 -3.28 2.87 0.0493 QPP4-1 vs P3 -6.00 3.83 2.21 -12.18 0.18 -2.71 3.93 0.0544 QPP4-2 vs P3 -35.00 4.07 2.35 -41.54 -28.46 -14.85 4.00 0.0001 97.70% vs 100% QPP4-3 vs P3 -14.00 4.97 2.87 -22.30 -5.70 -4.88 3.63 0.0104 QPP2-1 vs P2 -24.83 11.10 6.41 -46.12 -3.55 -3.88 2.79 0.0345 QPP2-2 vs P2 -26.33 5.23 3.02 -35.76 -16.91 -8.72 3.11 0.0027 94.12% vs 100% QPP2-3 vs P2 -10.00 7.00 4.04 -21.31 1.31 -2.47 3.92 0.0699 QPP6-1 vs P3 -69.00 6.79 3.92 -79.90 -58.10 -17.62 3.97 6.43E-05 QPP6-2 vs P3 -50.67 5.42 3.13 -60.03 -41.31 -16.20 3.37 0.0002 100% vs 100% QPP6-3 vs P3 -75.67 5.42 3.13 -85.03 -66.31 -24.20 3.37 6.72E-05 QPP5-1 vs P5 -52.67 6.81 3.93 -63.60 -41.73 -13.401 3.9751 0.0002 QPP5-2 vs P5 -49.00 5.11 2.95 -60.81 -37.19 -16.644 2.1597 0.0026 90.68% vs 100% QPP5-3 vs P5 -53.33 5.23 3.02 -64.56 -42.11 -17.669 2.3701 0.0014 QPP3-1 vs P9 -18.00 4.76 2.75 -26.25 -9.75 -6.5485 3.3483 0.0051 QPP3-2 vs P9 -27.33 3.22 1.86 -32.59 -22.08 -14.728 3.8059 0.0002 94.23% vs 100% QPP3-3 vs P9 -25.00 4.76 2.75 -33.25 -16.75 -9.0951 3.3483 0.0018
Note. QPP1 (introgression from F1 cross CML154Q x P3), QPP2 (introgression from F1 cross
CML154Q x P2), QPP3 (introgression from F1 cross CML 154Q x P9), QPP4 (K0326Y x P3),
QPP5 (K0326Y x P5), QPP6 (Tx807 x P3) and popcorn lines were used for small-scale popping analysis. Three different BC2F5 populations (indicated as QPP-1, -2 and -3) were used for each introgression to show variation for popping volume between BC2F5 ears. Three pools of kernels (3.33g each) were prepared from each ear as replicates. The total volume measured was recorded for comparison. Comparison was carried out using two sample t-test (Welch’s t test). Highlighted in bold are the ones without significant difference in popping volume (α =
0.05) which are QPP1-2, QPP4-1, QPP2-3. Welch’s t-test adjusts the number of degree of freedom when the variances are thought not to be equal to each other. The average differences between QPP replicates and corresponding popcorn parental lines were shown in the Mean
Difference column. The standard deviation of the average difference was shown in column
155 Std. Deviation Difference. Std. Error of the average difference was shown in column Std. Error
Difference. The lower and upper boundaries of the 95% confidence interval of the true mean
difference was shown in column 95% of Confidence Interval of the Difference. The t statistic
was obtained by dividing the mean difference by its standard error. Probability of obtaining a
t statistic with its absolute value being equal to or greater than the obtained statistic was
indicated in the Sig. (2-tailed) column. In the Percentages of popped kernels column, ratio of
popped kernels were calculated as “Sum of popped kernels in three replicates” divided by
“Sum of kernels in three replicates prepared for popping”.
156 REFERENCES
Angelovici, R., Lipka, A.E., Deason, N., Gonzalez-Jorge, S., Lin, H., Cepela, J., Buell,
R., Gore, M.A., and DellaPenna, D. 2013. Genome-wide analysis of branched-
chain amino acid levels in arabidopsis seeds. The Plant Cell, 25(12), 4827-
4843.
Fountoulakis, M., and Lahm, H.W. 1998. Hydrolysis and amino acid composition
analysis of proteins. Journal of Chromatography A, 826(2), 109-134.
Hunter, B.G., Beatty, M.K., Singletary, G.W., Hamaker, B.R., Dilkes, B.P.,
Larkins, B.A., and Jung, R. 2002. Maize opaque endosperm mutations create
extensive changes in patterns of gene expression. The Plant Cell, 14(10), 2591-
2612.
157
CHAPTER 4
DISCUSSION ON THE IMPEDIMENTS OF QPP
BREEDING AND SOLUTIONS, ITS PROSPECTIVE AND
FUTURE PLANS
158 1. Discussion on the impediments of QPP breeding and solutions
Beside the unclarity about the modifier QTLs which entails difficulties in QPM
conversion studies, another drawback of QPM conversion can be poor seed set
throughout selection which can lead to the loss of targeted crosses (Kostadinovic
et al., 2016). This phenomenon may be attributable to incompatibility between
pollen and style. In our study, this was problematic in the BC2F2. Although many
crosses seemed promising for modifier transfer with significant p-values (p < 0.05)
in F2 populations, because of poor seed set in the BC2F2, we were not able to select enough Type II and Type III opaque BC2F2 individuals for some crosses (Table 2
in Chapter 2).
Reduction of popping quality is commonly observed in dent corn by popcorn
populations and thus is a major concern (Dhliwayo, 2008; Dofing et al., 1991; Li
et al., 2009; Lu et al., 2003; Robbins and Ashman, 1984; Yu et al., 2006). However,
studies confirmed that PEV could be partially recovered by backcrossing to
popcorn parents while comparing the popping volume of F1, BC1 and BC2
populations (Crumbaker et al., 1949; Johnson and Eldredge, 1953). The
complexity of the genetic loci contributing to PEV makes its maintenance
challenging. Previously, it was reported that two generation backcrossing was
sufficient to recover acceptable PEV. The characteristic distinction in shape and
size of kernels between popcorn and dent corn can be used for selection and
accelerated recovery of popcorn genome. Using two generations of backcrossing
combined with subsequent phenotypic selection for both kernel shape and ear
159 characteristics, we selected inbred lines with comparable PEV to the original
popcorn parents. Variation between BC2F5 populations indicated that there is scope for further selection of PEV. Besides recovery of popping, for introgression of dent traits into commercial lines with cross-incompatibility, maintaining dent sterility is essential to maintain popcorn’s genetic isolation and ability to be grown in proximity to GM corn. We initiated F1 crosses unidirectionally using popcorn
parental lines as males and then used the F1 and backcross generations as males to cross to popcorn females in subsequent generations. This ensured the maintenance of dent sterility since only pollen bearing the trait of dent-sterility could successfully pollinate the popcorn recurrent parent. Introgression of opaque 2 relies on opaque 2 in-gene SSR markers. Genotyping of these markers requires high
concentration agarose gels which is time-consuming and of low resolution.
Furthermore, no polymorphic marker was available for certain crosses initiated in
our breeding program. For these certain crosses, we selfed all BC2 individuals
planted and later determined whether the BC2 is heterozygous or not by evaluating the modification of the resultant BC2F2 ear. This was all relying on phenotypic
selection in early selfing generations (BC2F2 and BC2F3) after backcrossing and
SDS-PAGE. This confirmed that SDS-PAGE is enough for QPM conversion. The reason why we still need Opaque2 polymorphic markers is that we are carrying multiple introgressions simultaneously. It would have been more work without using Opaque 2 in-gene markers. Newer generation of markers such as SNP would also be considered in the future (Tandzi et al., 2017). Mapping studies of endosperm modification modifiers depend heavily on the phenotyping data from
160 evaluation of endosperm modification. Currently the scale is based entirely on
subjective visualization of the kernels on light box which is not of high resolution.
In the future, better and more accurate phenotyping methods is expected.
2. QPP prospective and future plans
Malnutrition has been a major health problem across developing countries (Müller
and Krawinkel, 2005). Popcorn enjoyed nearly a constant increase in sales during the
second half of the twentieth century (Sweley et al., 2013). This was due to significant
advancements in popcorn quality traits such as maximizing popping expansion
volume (PEV) and percentage popped kernels as well as flake type and quality as
well as the development of microwave popcorn. Since this time, few technological advances have been introduced and sales of popcorn have plateaued. Strategies for invigorating the popcorn industry should be multipronged and involve agronomic
improvement and other aspects such as nutritional quality while considering consumer preferences. QPP, with its natural opaque-2 and modifier alleles, and subsequent production of high yielding hybrids is one way to answer this call.
Awareness of food nutrition is relatively high, (as it is deemed as important by
59.9% of U.S. adults, second to taste (77%)) according to the National Health and
Nutrition Examination Survey (Aggarwal et al., 2016). The development and utilization of more nutritious popcorn could have both economic and humanitarian application. The focus on popping-related traits rather than plant agronomic traits
has resulted in much less agronomic improvement than in dent corn. For instance,
yields of popcorn are less than dent corn (Ziegler, 2000). The agronomic
161 superiority of dent corn has potential to contribute to popcorn improvements and
led to several studies using crosses between dent corn and popcorn. As one way to
avert protein malnutrition, many QPM conversion studies were carried out
previously for dent corn (Babu et al., 2005; Gupta et al., 2009, Gupta et al., 2013;
Jompuk et al., 2011; Krishna et al., 2017; Sofi et al., 2009; Surender et al., 2017).
Common challenges in QPM conversion programs included incomplete recovery
of vitreous endosperm (modification), discontinuation of certain crosses from poor
seed set and failure to improve lysine and tryptophan content (Kostadinovic et al.,
2016; Moro et al., 1996; Prasanna et al., 2001; Vivek et al., 2008). Since popcorn
kernels naturally have a very high proportion of vitreous endosperm (Figure 3 in
Chapter 3), and this is the kernel region in which starch can melt during the
popping process, complete endosperm modification is likely of paramount
importance in QPM conversion into a popcorn background. o2 mutants without
modification are associated with soft kernels and loss of popping as observed in
several opaque-2 introgression studies (Adunola, 2017; Zhou et al., 2016). By
evaluation of modifier transfer at the F2 generation, we targeted crosses at an early
stage, that had greater potential for later full restoration of vitreous, and likely
poppable kernels. For these promising crosses, selection for endosperm
modification was carried out on an individual basis in BC2F2 generation (Type II and Type III opaque BC2F2 individuals) and then on a whole ear basis in later
generations (BC2F3, BC2F4, BC2F5). Selection of Type II and Type III opaque kernels at the BC2F2 population is key step. The ongoing six BC2F4 QPP
introgressions had, on average, four promising BC2F2 populations segregating for
162 Type I through Type IV opaqueness whereas the discontinued crosses were the
ones which showed close to 25% Type IV-V (unmodified/opaque) kernels
segregating in BC2F2 populations.
The 0707-1 marker recently reported for detection the presence of duplication event at the major QTL 27kD γ-zein (Liu et al., 2016) can be applied in early stages of back-crossing generations to help in selection of promising BC2F2 populations.
The increased uniformity in endosperm modification observed from BC2F2 to
BC2F4 indicated that the modifiers likely reached homozygosity during this process. In BC2F4 populations tested with SDS-PAGE, the uniformly increased level of 27kD γ-zein protein likely indicates that the allelic composition 27kD γ- zein locus reached homozygosity for the duplicated allele.
Previously, failure to improve lysine content was reported despite successful introgression of o2 and modifier genes (Babu et al., 2005; Moro et al., 1996; Vivek et al., 2008; Zhao et al., 2012). This problem was not observed in any of our six
BC2F4 introgressions and the increases in both protein-bound lysine and free lysine
were significant for all BC2F4 QPP introgressions. The process of proteome
rebalancing means that decrease of lysine devoid zein proteins will result in
increased accumulation of lysine containing non-zeins (Wu and Messing, 2014).
In addition to this global increase in non-zeins, it was shown that the most elevated
non-zein proteins are enriched in lysine (Morton et al., 2015). For QPP to be useful,
it is important that the kernels do not have reduced total protein. No total protein
reductions were observed compared with either the popcorn or QPM parents and
163 in fact, a significant increase in total protein is shown in two of our introgressions
(Figure 7 in Chapter 3). Most free amino acids are increased in QPP (Table 1 in
Chapter 3) and this may be partly as a result of reduced incorporation into zeins.
Substantial increases in the levels of most free amino acids were observed in several types of maize mutants with reduced zeins (Galili and Amir, 2013). However, since zeins do not contain lysine, this does not explain the increase in free lysine. The free amino acids increase was initially demonstrated in the o2 mutants. In addition to zein genes, O2 also regulates other genes including one encoding a lysine catabolic enzyme lysine-ketoglutarate reductase / saccharopine dehydrogenase (LKR-SDH)
(Brochetto-Braga et al., 1992; Kemper et al., 1999; Li et al., 2015). The resulting reduction in lysine degradation contributes to the observed free-lysine increase in o2 mutant. PCA of protein bound amino acids indicated that the QPP introgressions grouped with QPMs but were distant from the group of wild type popcorn parents. Amino acids showing uniform increase (Asx, His, Lys, Arg) or decrease (Met, Ser, Glx, Ala, Ile, Leu, Phe) between all o2 (QPM parents, QPPs and B73 o2) and all wild type germplasms (popcorn parents and B73) in this study were consistent with the comparison of amino acid composition between a wild type (W64A) and its isogenic line W64Ao2 (Hunter et al., 2002) (Figure 6 in
Chapter 3). In the PCA analysis of free amino acids, wild type (popcorn parents,
B73) were grouped together, whereas no obvious pattern were observed across different o2 mutants (QPM parents, QPPs and B73o2) (Figure 5 in Chapter 3).
The strategy outlined here for introgression of dent corn traits into popcorn backgrounds brings together selection for QPM (o2 and modifiers) and for popcorn
164 characteristics (dent sterility, kernel shape, high proportion of vitreous endosperm), resulting in the first demonstration that development of QPP is achievable. The reduced popping in some lines indicated that further selection for endosperm modification is required. Both selfing and backcrossing for increased popcorn genomic background are planned out.
Generation and evaluation of QPP hybrids
Previously, efforts were made toward QPM hybrids. Both general and specific combining ability for grain yield, protein concentration in grain, tryptophan concentration in grain and in protein were assessed for various tropical, late- maturing and white-grained QPM lines (Pixley and Bjarnason, 1993). Combining ability study between different QPMs was carried out (Vasal et al., 1993). In our study, the six independent BC2F4 introgressions between three QPMs and four popcorn parents, all carrying the homozygous o2 allele, gives excellent scope for
QPP hybrid production and testing which has begun. Performance of hybrid including agronomic traits and kernel vitreousness and popping ability will be evaluated.
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and Jung, R. 2002. Maize opaque endosperm mutations create extensive changes in
patterns of gene expression. The Plant Cell, 14(10), 2591-2612.
Johnson, I.J., and Eldredge, J.C. 1953. Performance of recovered popcorn inbred lines derived
from outcrosses to dent corn. Agronomy Journal, 45(3), 105-110.
Jompuk, C., Cheuchart, P., Jompuk, P., and Apisitwanich, S. 2011. Improved tryptophan
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Kemper, E.L., Neto, G.C., Papes, F., Moraes, K.C.M., Leite, A., and Arruda, P. 1999. The role
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CHAPTER 5
APPENDIX
171 1. R codes for data analysis and presentation
1.1 PCA analysis of protein-bound amino acids (PBAA)
R codes used for PCA analysis of PBAA measurements are as follows. rm(list=ls()) setwd("~/Desktop") %library(gdata) # load gdata package paa = read.xls("amino acid_paa.xlsx") # read from first sheet paa is.data.frame(paa) #[1] TRUE library(ggplot2) data_summary <- function(data, varname, groupnames){ require(plyr) summary_func <- function(x, col){ c(mean = mean(x[[col]], na.rm=TRUE), sd = sd(x[[col]], na.rm=TRUE)) } data_sum<-ddply(data, groupnames, .fun=summary_func, varname) data_sum <- rename(data_sum, c("mean" = varname)) return(data_sum) } paa1<-data_summary(paa,varname="lysine",groupnames="genotype") orderedgenotype <- factor(paa1$genotype, levels=c("1.CML154Q", "2.K0326Y", "3.Tx807","4.P3","5.P2","6.P5","7.P9","8.QPP1","9.QPP2","10.QPP3","11.QPP4","12.Q PP5","13.QPP6")) jpeg("ProteinBoundLysine.jpg", width=6, height=5, units="in", res=500) p<- ggplot(paa1, aes(x=orderedgenotype, y=lysine,width=0.6))+ labs(x="Genotype")+ scale_y_continuous(limits=c(0,0.53),expand=c(0,0)) + scale_x_discrete("Genotype",labels=c("CML154Q", "K0326Y", "Tx807","P3","P2","P5","P9","QPP1","QPP2","QPP3","QPP4","QPP5","QPP6")) + theme_bw(base_size = 10) + theme(axis.text.x=element_text(angle=180,hjust=1), axis.line = element_line(colour = "black"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank())+
172 geom_bar(stat="identity", fill=c("dimgrey","dimgrey","dimgrey","dimgrey","dimgrey","dimgrey","dimgrey","dimg rey","dimgrey","dimgrey","dimgrey","dimgrey","dimgrey"),color=c(rep("black",13)),pos ition=position_dodge(width=0.7),width = 0.2)+ geom_errorbar(aes(ymin=lysine-sd, ymax=lysine+sd), width=.2,position=position_dodge(.9)) print(p) p+labs(title="Protein-bound Lysine per genotype")+labs(y = "Protein-bound lysine (W/W%)")+ theme(text = element_text(size=20),axis.text.x = element_text(angle = 60, hjust = 1)) dev.off()
1.2 PCA analysis of free amino acids (FAA)
R codes used for PCA analysis of FAA measurements are as follows. rm(list=ls()) setwd("~/Desktop") library(gdata) # load gdata package faa = read.xls("amino acid_faa.xlsx") # read from first sheet faa is.data.frame(faa) #[1] TRUE library(ggplot2) data_summary <- function(data, varname, groupnames){ require(plyr) summary_func <- function(x, col){ c(mean = mean(x[[col]], na.rm=TRUE), sd = sd(x[[col]], na.rm=TRUE)) } data_sum<-ddply(data, groupnames, .fun=summary_func, varname) data_sum <- rename(data_sum, c("mean" = varname)) return(data_sum) } faa1<-data_summary(faa,varname="lysine",groupnames="genotype") orderedgenotype <- factor(faa1$genotype, levels=c("1.CML154Q", "2.K0326Y", "3.Tx807","4.P3","5.P2","6.P5","7.P9","8.QPP1","9.QPP2","10.QPP3","11.QPP4","12.Q PP5","13.QPP6")) jpeg("FreeLysine.jpg", width=6, height=5, units="in", res=500) p<- ggplot(faa1, aes(x=orderedgenotype, y=lysine,width=0.6)) +
173 labs(x="Genotype")+ scale_y_continuous(limits=c(0,0.031),expand=c(0,0)) + scale_x_discrete("Genotype",labels=c("CML154Q", "K0326Y", "Tx807","P3","P2","P5","P9","QPP1","QPP2","QPP3","QPP4","QPP5","QPP6")) + theme_bw(base_size = 10) + theme(axis.text.x=element_text(angle=180,hjust=1), axis.line = element_line(colour = "black"), panel.grid.major = element_blank(), panel.grid.minor = element_blank(), panel.border = element_blank(), panel.background = element_blank())+ geom_bar(stat="identity", fill=c("dimgrey","dimgrey","dimgrey","dimgrey","dimgrey","dimgrey","dimgrey","dimg rey","dimgrey","dimgrey","dimgrey","dimgrey","dimgrey"),color=c(rep("black",13)),pos ition=position_dodge(width=0.7),width = 0.2)+ geom_errorbar(aes(ymin=lysine-sd, ymax=lysine+sd), width=.2, position=position_dodge(.9)) print(p)
p+labs(title="Free Lysine per genotype")+labs(y = "Free lysine (W/W%)")+ theme(text=element_text(size=20),axis.text.x = element_text(angle = 60, hjust = 1)) dev.off()
1.3 Comparison of log2 value of ratio (o2 / wild type ratio) for 15 amino acids using protein-bound amino acid (PBAA) measurements
R codes used for comparison of log2 value of ratio (o2 / wild type ratio) for 15
amino acids using protein-bound amino acid (PBAA) measurements are as follows.
rm(list=ls()) setwd("~/Desktop") aminoacids_15<-read.csv("raw_ratio_o2bywt.csv",header = TRUE,row.names='ID',col.names = c("ID","ratio","Asx","Met","Thr","Ser","Glx","Pro","Gly","Ala","Val","Ile","Leu","Phe", "His","Lys","Arg"),sep=",") dim(aminoacids_15) aminoacids15<-aminoacids_15[,(-1)] head(aminoacids15) aminoacids15<-as.matrix(aminoacids15)
174 dim(aminoacids15)<-c(dim(aminoacids15)[1]*dim(aminoacids15)[2],1) dim(aminoacids15) v<-as.vector(aminoacids15) length(v) is.vector(v) Log2_ratio<-log2(v) library(ggplot2) # create a dataset aminoacids<- rep(c("Asx","Met","Thr","Ser","Glx","Pro","Gly","Ala","Val","Ile","Leu","Phe","His"," Lys","Arg"),each=14) ratio_of_comparison<-c(rep(c("hunter o2 by wt","B73 o2 by wt","Q1xP1 Introgression by P1","Q1 by P1","Q1xP2 Introgression by P2","Q1 by P2","Q1xP4 Introgression by P4","Q1 by P4","Q2xP1 Introgression by P1","Q2 by P1","Q2xP3 Introgression by P3","Q2 by P3","Q3xP2 Introgression by P2","Q3 by P2"),15)) data=data.frame(aminoacids,ratio_of_comparison,Log2_ratio) data order<-as.factor(rep(c(8,15,1,5,7,13,10,11,14,2,12,6,4,3,9),14)) data=cbind(data,order) levels(data$order)<- c("Asx","Met","Thr","Ser","Glx","Pro","Gly","Ala","Val","Ile","Leu","Phe","His","Lys", "Arg") # Grouped library(RColorBrewer) data$ratio_of_comparison<-factor(data$ratio_of_comparison,levels=c("hunter o2 by wt","B73 o2 by wt","Q1xP1 Introgression by P1","Q1 by P1","Q1xP2 Introgression by P2","Q1 by P2","Q1xP4 Introgression by P4","Q1 by P4","Q2xP1 Introgression by P1","Q2 by P1","Q2xP3 Introgression by P3","Q2 by P3","Q3xP2 Introgression by P2","Q3 by P2")) data$aminoacids<- factor(data$aminoacids,levels=c("Asx","Met","Thr","Ser","Glx","Pro","Gly","Ala","Val ","Ile","Leu","Phe","His","Lys","Arg")) jpeg("log2 Value of ration for all 15 amino acids.jpg", width=10, height=5, units="in", res=500) ggplot(data, aes(fill=ratio_of_comparison, y=Log2_ratio, x=aminoacids,width=0.8)) + geom_bar(position=position_dodge(width=0.7), stat="identity", width =1)+ theme(legend.background = element_rect(fill = "transparent"), #legend.box.background = element_rect(fill = "transparent"), panel.grid.minor = element_blank(), panel.grid.major = element_blank() #panel.background = element_rect(fill = "transparent",colour = NA) #plot.background = element_rect(fill = "transparent",colour = NA) )+ geom_hline(yintercept=0,color='grey60',size=0.1) dev.off()
175
1.4 Comparison of the relative concentration of total protein
R codes used for comparison of relative concentration of total protein are as follows. rm(list=ls()) library(gdata) setwd("~/Desktop") rela_con<-read.xls("concentration comparison for total protein between QPPs and corresponding popcorn parental lines.xlsx") is.data.frame(rela_con) t.test(rela_con[rela_con$comparison=="QPP1",2],rela_con[rela_con$comparison=="P3/ QPP1",2]) t.test(rela_con[rela_con$comparison=="QPP2",2],rela_con[rela_con$comparison=="P2/ QPP2",2]) t.test(rela_con[rela_con$comparison=="QPP3",2],rela_con[rela_con$comparison=="P9/ QPP3",2]) t.test(rela_con[rela_con$comparison=="QPP4",2],rela_con[rela_con$comparison=="P3/ QPP4",2]) t.test(rela_con[rela_con$comparison=="QPP5",2],rela_con[rela_con$comparison=="P5/ QPP5",2]) t.test(rela_con[rela_con$comparison=="QPP6",2],rela_con[rela_con$comparison=="P2/ QPP6",2]) library(ggplot2) #install.packages("ggsignif") library(ggsignif) data_summary <- function(data, varname, groupnames){ require(plyr) summary_func <- function(x, col){ c(mean = mean(x[[col]], na.rm=TRUE), sd = sd(x[[col]], na.rm=TRUE)) } data_sum<-ddply(data, groupnames, .fun=summary_func, varname) data_sum <- rename(data_sum, c("mean" = varname)) return(data_sum) } rela_con1<-data_summary(rela_con,varname="ratio",groupnames="comparison") orderedgenotype <- factor(rela_con1$comparison, levels=c("QPP1", "P3/QPP1","QPP2","P2/QPP2","QPP3","P9/QPP3","QPP4","P3/QPP4","QPP5","P5/QP P5","QPP6","P2/QPP6"))
176 jpeg("Comparison of the relative concentration of total protein.jpg", width=8.5, height=4, units="in", res=500) p<- ggplot(rela_con1, aes(x=orderedgenotype, y=ratio,fill=orderedgenotype)) + labs(x=NULL)+ scale_y_continuous(limits=c(0,1.5),expand=c(0,0)) + geom_bar(stat="identity",colour=c("black"),position=position_dodge(width=0.3),width = 0.78,size=.4)+scale_fill_manual(values = c("gray25","gray56","gray25","gray56","gray25","gray56","gray25","gray56","gray25"," gray56","gray25","gray56"))+ geom_errorbar(aes(ymin=ratio-sd, ymax=ratio+sd),color="black", width=.2, position=position_dodge(.9))+ geom_signif(comparisons=list(c("QPP1","P3/QPP1")),annotations = "Sig. p=0.03974",map_signif_level = TRUE,y_position = 1.2)+ geom_signif(comparisons=list(c("QPP4","P3/QPP4")),annotations = "Sig. p=0.0166",map_signif_level = TRUE,y_position=1.4) print(p) p+labs(title="Comparison of the relative concentration of total protein")+ theme(legend.position="none",axis.text.x = element_text(angle = 60, hjust = 1)) dev.off()
1.5 Popping volume comparison
R codes used for popping volume comparison are as follows. rm(list=ls()) setwd("~/Desktop") library(gdata) # load gdata package pv = read.xls("popping volume.xlsx") # read from first sheet pv is.data.frame(pv) #[1] TRUE library(ggplot2) #install.packages("ggsignif") library(ggsignif) data_summary <- function(data, varname, groupnames){ require(plyr) summary_func <- function(x, col){ c(mean = mean(x[[col]], na.rm=TRUE), sd = sd(x[[col]], na.rm=TRUE)) } data_sum<-ddply(data, groupnames, .fun=summary_func,
177 varname) data_sum <- rename(data_sum, c("mean" = varname)) return(data_sum) } pv1<-data_summary(pv,varname="Popping_volume",groupnames="genotype") pvalue<-v() is.vector(pvalue) #independent two sample t test (unpaired), by default, t.test does not assume equal variances, instead of student's t-test, #it used the Welch t-test by default. In Welch t-test, df=17.776, because of the adjustment for unequal variance. # To use student's t-test, set var.equal=TRUE. t.test(pv[pv$genotype=="QPP1-1",2],pv[pv$genotype=="P3",2]) t.test(pv[pv$genotype=="QPP1-2",2],pv[pv$genotype=="P3",2]) #NS t.test(pv[pv$genotype=="QPP1-3",2],pv[pv$genotype=="P3",2]) t.test(pv[pv$genotype=="QPP4-1",2],pv[pv$genotype=="P3",2]) #NS t.test(pv[pv$genotype=="QPP4-2",2],pv[pv$genotype=="P3",2]) t.test(pv[pv$genotype=="QPP4-3",2],pv[pv$genotype=="P3",2]) t.test(pv[pv$genotype=="QPP2-1",2],pv[pv$genotype=="P2",2]) t.test(pv[pv$genotype=="QPP2-2",2],pv[pv$genotype=="P2",2]) t.test(pv[pv$genotype=="QPP2-3",2],pv[pv$genotype=="P2",2]) #NS t.test(pv[pv$genotype=="QPP6-1",2],pv[pv$genotype=="P2",2]) t.test(pv[pv$genotype=="QPP6-2",2],pv[pv$genotype=="P2",2]) t.test(pv[pv$genotype=="QPP6-3",2],pv[pv$genotype=="P2",2]) t.test(pv[pv$genotype=="QPP5-1",2],pv[pv$genotype=="P5",2]) t.test(pv[pv$genotype=="QPP5-2",2],pv[pv$genotype=="P5",2]) t.test(pv[pv$genotype=="QPP5-3",2],pv[pv$genotype=="P5",2]) t.test(pv[pv$genotype=="QPP3-1",2],pv[pv$genotype=="P9",2]) t.test(pv[pv$genotype=="QPP3-2",2],pv[pv$genotype=="P9",2]) t.test(pv[pv$genotype=="QPP3-3",2],pv[pv$genotype=="P9",2]) orderedgenotype <- factor(pv1$genotype, levels=c("P3", "QPP1-1","QPP1-2","QPP1- 3","QPP4-1","QPP4-2","QPP4-3","P2","QPP2-1","QPP2-2","QPP2-3","QPP6-1","QPP6- 2","QPP6-3","P5","QPP5-1","QPP5-2","QPP5-3","P9","QPP3-1","QPP3-2","QPP3-3")) jpeg("popingvol comparison.jpg", width=8.5, height=4, units="in", res=500) p<- ggplot(pv1, aes(x=orderedgenotype, y=Popping_volume)) + labs(x=NULL)+ scale_y_continuous(limits=c(0,160),expand=c(0,0)) + geom_bar(stat="identity",fill=c("blue","cornflowerblue","cornflowerblue","cornflowerbl ue","deepskyblue","deepskyblue","deepskyblue","springgreen4","green3","green3","gree n3","darkseagreen2","darkseagreen2","darkseagreen2","lightgoldenrod3","lightgoldenrod 1","lightgoldenrod1","lightgoldenrod1","snow4","snow3","snow3","snow3"),color=c(rep ("black",22)),position=position_dodge(width=0.7),width = 1)+ geom_errorbar(aes(ymin=Popping_volume-sd, ymax=Popping_volume+sd), width=.2, position=position_dodge(.9))+ geom_signif(comparisons=list(c("P3","QPP1-2")),annotations = "NS, p=0.6495",map_signif_level = TRUE,y_position = 132.4,xmin=c())+
178 geom_signif(comparisons=list(c("P3","QPP4-1")),annotations = "NS, p=0.05441",map_signif_level = TRUE,y_position=145.6)+ geom_signif(comparisons=list(c("P2","QPP2-3")),annotations = "NS, p=0.06991",map_signif_level = TRUE,y_position=108.6) print(p) p+labs(title="Comparison of popping volume (ml)")+ theme(axis.text.x = element_text(angle = 60, hjust = 1)) dev.off()