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Jaecklein 1
The Florida State University
College of Arts and Sciences
Characterization of DNA methylation of the endogenous gene,
Tgs5, in tgr1 Zea mays mutants
Eleni E. Jaecklein
A Thesis submitted to the
Department of Biology
in partial fulfillment of
requirements for graduation with
Honors in the Major
Degree Awarded:
Spring Semester, 2016
Jaecklein 2
Dr. Karen McGinnis
Thesis Director
Dr. Lisa Eckel
Outside Committee Member
Dr. Thomas Keller
Committee Member
Jaecklein 3
Eukaryotic exposure to transposable elements and exogenous DNA necessitates the existence
of mechanisms devoted to maintaining genome stability. Transcriptional gene silencing is
associated with epigenetic modifications such as DNA methylation, histone modification, and
changes in chromatin structure. In plants, an RNA-dependent DNA Methylation (RdDM)
pathway represses the expression of transposable elements, repetitive sequences, and
transgenes via RNA directed cytosine methylation. In Zea mays, the RdDM pathway stably,
transcriptionally silences the b1 genomic transgene, which codes for a transcription factor
involved in the production of a dark purple pigment called anthocyanin. Mutants of RdDM are
easily identified using this transgene system, because they display dark purple pigmentation. In
this investigation we sought to assess the RdDM pathway’s involvement in regulating
endogenous loci in Zea mays using the RdDM mutant, tgr1. Several endogenous genes were
previously identified as potential RdDM targets based on proximity to transposable elements,
changes in gene expression in RdDM mutants, and changes in 24-nt small interfering RNAs
(siRNAs). One of these genes was selected for analysis, Tgs5. Bisulfite conversion was used to
evaluate cytosine methylation in the promoter region of Tgs5 in wild type and mutant plants.
However, sequencing data revealed non-specific amplification and no conclusions could be
drawn about Tgs5 or other endogenous RdDM targets.
Jaecklein 4
All living organisms require transcriptional regulation of gene expression. Transcriptional
regulation occurs on multiple levels of genome organization and is associated with the
trademarks of epigenetic regulation, namely histone modification and DNA methylation
(Goldberg et al. 2007; Meyer 2000). These epigenetic modifications lead to changes in
chromatin structure, which impacts the ability of transcription factors and other mediators of
transcription to access the DNA. Consequently, alterations in chromatin structure ultimately
influences gene expression and phenotype. A change in gene expression and phenotype caused
by epigenetic regulation occurs without altering the DNA sequence, but is still heritable,
mitotically and, in some cases, meiotically (Deans et al. 2015; Goldberg et al. 2007; Jarillo et al.
2009). Methylation of cytosine residues in repetitive sequences, transposable elements, and in
gene promoter regions has been linked to transcriptional gene silencing (Lee et al. 2010; Mette
et al. 2000). Transcriptional gene silencing (TGS) occurs by suppressing transcription; however,
gene silencing can also occur post-transcriptionally (PTGS) via mRNA degradation (Vaucheret et
al. 2001). TGS is an adaptive mechanism employed by many eukaryotes, presumably to cope
with genome alternation incurred by exposure to transposable elements, transgenes, and
exogenous viral DNA (reviewed by Kooter et al. 1999). This form of epigenetic regulation
appears to be conserved in both plants and animals; however, they differ in methylation
patterns (Lee et al. 2010).
Transposable elements comprise almost 85% of the maize genome, which makes Zea
mays an ideal model organism for studying TGS via DNA methylation (Baucom et al. 2009;
Schnable et al. 2009). In plants, methylation of the promoter region and subsequent gene
Jaecklein 5
silencing can be induced by double stranded RNAs (dsRNA) with sequences that are
homologous to the promoter region (Mette et al. 2000). The pathway responsible for this
epigenetic transcriptional gene silencing is known as the RNA-dependent DNA methylation
pathway or RdDM. RdDM relies on the coordination of many proteins and polymerases to
selectively methylate and silence regions of the maize genome (M. A. Matzke et al. 2015; M. A.
Matzke et al. 2014). Plant-specific polymerase IV creates a single stranded RNA (ssRNA)
sequence, which interacts with mediator of paramutation-1, MOP1, to create double stranded
(dsRNA). The dsRNA is chopped into 24-nt small interfering RNAs (siRNA), which guide silencing
machinery to the homologous target loci (M. Matzke et al. 2009; Simon et al. 2011; Zhang et al.
2011). RdDM has been extensively characterized in Arabidopsis; however, many maize RdDM
orthologs have been identified and associated with TGS at endogenous and exogenous loci
(reviewed by Arteaga-Vazquez et al. 2010).
In maize, the RdDM pathway is studied using a transgenic maize line (McGinnis et al.
2006). The b1 gene encodes a transcription factor that stimulates the expression of enzymes
involved in the production pathway of a dark purple pigment called anthocyanin in a tissue
specific manner (Chandler et al. 1989). Using biolistic particle bombardment, the b1 gene was
inserted in the Hi II maize genome, resulting in transgenic plants with active b1 genomic
transgene (BTG-active) having purple tissue (Selinger et al. 1998). The b1 genomic transgene
(BTG) also contains the 35S cauliflower mosaic virus (35S CaMV) promoter and the first intron
of alcohol dehydrogenase1 (adh1), which were included to enhance expression of the
transgene (Madzima et al. 2011). A line of plants was identified where BTG was stably,
transcriptionally silenced (BTG-silent) by the RdDM pathway, which resulted in green plants
Jaecklein 6
(McGinnis et al. 2006). Paramutation was eliminated as a potential source of silencing, because
the transgenic maize did not carry any b1 alleles or other sequences required for b1
paramutation, where paramutation is the trans interaction of two alleles that results in
alteration of the expression level of one allele in a meiotically heritable manner (Chandler
2010). The epigenetic transcriptional silencing of BTG occurs independently of paramutation
and is therefore dependent on the RdDM pathway (Madzima et al. 2011; McGinnis et al. 2006).
The green phenotype associated with BTG-silent individuals is a dominant trait and is referred
to as wild type. BTG-active is a recessive trait and homozygous recessive individuals are
referred to as mutants (McGinnis et al. 2006). Mutant individuals are easily identified due to
their purple phenotype, which is a result of BTG reactivation/ loss of silencing in the BTG-silent
line. BTG reactivation is associated with loss of cytosine methylation in the 35S CaMV promoter
region of the transgene (Madzima et al. 2011; McGinnis et al. 2006) (Figure 1A). Genes involved
in transcriptional gene silencing are denoted as tgr, for transgene-reactivated. BTG-silent has
been used to identify several tgr mutants involved in the RdDM pathway (tgr 1-6 and tgr 8-11),
which result in the reactivation of the transgene with homozygous recessive individuals
displaying the purple phenotype (Madzima et al. 2011). Tgr1 is believed to be the largest
subunit of polymerase IV and has also been identified as Mop3 in Zea mays (Sloan et al. 2014).
Homozygous mutants of tgr1/ mop3 display the purple phenotype accompanied
hypomethylation of the 35S CaMV BTG promoter region (Madzima et al. 2011)
Although endogenous RdDM targets have been identified in the Arabidopsis, such
analysis has not been completed in maize. Sixteen endogenous genes were characterized in
mop1-1 and tgr1-1 mutants as putative RdDM targets (Madzima et al. 2014). These genes were
Jaecklein 7
up regulated in both, mop1-1 and tgr1-1 mutants. Transposable elements (TEs) can influence
the expression of nearby genes especially if located +/- 1kb of the gene’s start or end, which is
associated with RNA-dependent de novo methylation, particularly CHH methylation (Gent et al.
2013; Lu et al. 2015). The transcriptional start sites (TSSs) of these sixteen genes were located
in close proximity, (+ /–1kb), to terminal inverted repeats (TIRs), which are conserved
sequences in class II transposable elements (Madzima et al. 2014). At least a twofold change in
homologous siRNAs were observed within 2kb downstream of the TSS in wild type (WT) and
mutant plants at these sixteen loci (Madzima et al. 2014). These characteristics are the
hallmarks of RdDM target loci, making these sixteen genes ideal locations to analyze
endogenous RdDM targets in Zea mays. These sixteen genes are referred to as Tgs1-16, for
transcriptionally gene silenced.
This investigation seeks to determine RdDM’s involvement in regulating the
endogenous gene, Tgs5, by characterizing DNA methylation in the promoter region. This study
tested the hypothesis that the promoter region of Tgs5 will be hypomethylated in the RdDM
mutant, tgr1, especially CHH methylation if RdDM is responsible for regulating the expression of
this endogenous gene. DNA extracted from the young leaf tissue of three tgr1 mutant plants
(P1, P2, P3) and three tgr1 wild type plants (G1, G2, G3) was analyzed using bisulfite conversion
and DNA sequencing.
Jaecklein 8
Experimental Design
Seed Selection and Genetic Background
All plants used in this investigation were from the b1 genomic transgenic (BTG) line
where BTG is heritably, stably, transcriptionally silenced as mentioned previously (McGinnis et
al. 2006) which had been crossed with mutagenized pollen to generate tgr mutants (Madzima
et al., 2011). Sixty transgenic seeds from a self-crossed, green, heterozygous tgr1 individual
were planted in the greenhouse. The seeds were expected to yield green homozygous
dominant/ heterozygous individuals and purple homozygous mutant individuals in a 3:1 ratio
(Figure 2).
Tissue Collection
After three and a half weeks of growth, tissue was collected from the plants.
Approximately six inches of young, leaf tissue were cut from the seventh leaf of each plant,
counting from the base up. The tissue was immediately wrapped in aluminum foil, immersed in
liquid nitrogen, and stored a -80°C for preservation. Plants were continually monitored for
immature ear growth; however, low yield in wild type plants prevented tissue collection.
DNA Extraction
Preserved leaf tissue from three tgr1 mutant individuals and three tgr1 wild type
individuals were ground into a powder using liquid nitrogen and a mortar and pestle.
Approximately 2 mL of powered tissue was collected in a 15mL conical tube. Genomic DNA
(gDNA) was then extracted according to the Madzima protocol for phase extraction using
Jaecklein 9
chloroform. The DNA pellets were re-suspended and concentrations were quantified using
NanoDrop. After RNase treatment the DNA was ready for bisulfite conversion.
Bisulfite Conversion
Bisulfite conversion is a simple acid-catalyzed reaction that converts unmethylated
cytosine into uracil, leaving methylated cytosine untouched. After a series of amplification steps
the uracil is converted into a thymine, thus altering the DNA sequence. Bisulfite conversion was
performed on 300ng of RNase treated gDNA from three tgr1 mutants and three tgr1 wild types
using the EZ DNA Methylation-Gold Kit from Zymo Research, according to the manufacturer’s
concentrations were quantified using NanoDrop.
Control and Primer Design
An area with a known methylation pattern was used as the control to ensure complete
conversion. The adh1 intron in the promoter region of the BTG transgene is consistently
unmethylated in both BTG-silent (wild type) and BTG-active (mutant) plants (Madzima et al.
2011). The 35S CaMV region of the BTG promoter is methylated in BTG-silent lines and
unmethylated in BTG-active lines (Madzima et al. 2011)(Figure A). A region incorporating a
portion of the 35S CaMV promoter and adh1 intron was used as the control region because it
displays a clear methylation pattern that is consistent in wild types and mutants. The 35S CaMV
promoter and adh1 intron was amplified and sequenced before continuing with Tgs5
Because the bisulfite conversion alters the DNA sequence, degenerate primers must be
used with converted DNA. Degenerate primers were designed using Kismeth, the plant bisulfite
Jaecklein 10
sequencing primer design program (Gruntman et al. 2008). The R refers to a purine and Y refers
to a pyrimidine.
Degenerate Primers:
35S CaMV Promoter and adh1 intron
Primer efficiency was confirmed with polymerase chain reaction (PCR) and gel electrophoresis,
using the following PCR conditions for the 35sCaMV promoter/ adh1 intron region: 95°C for 3
minutes (1x); 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds (35x); 72°C for 7
minutes. Conditions for Tgs5 were as follows: 95°C for 3 minutes (1x); 95°C for 30 seconds, 53°C
for 30 seconds, 72°C for 30 seconds (35x); 72°C for 7 minutes PCR product was stored at 4°C
until run on a 1% agarose gel at 140V for 30 minutes. Product size was confirmed using
ethidium bromide and UV imagining. The 35sCaMV promoter/ adh1 intron region is 410 base
pairs (bp) and the Tgs5 promoter region amplified is 291 bp. Using bisulfite converted DNA
from the three mutant and three wild type plants, a PCR reaction was conducted using the
same parameters outlined above. PCR product was purified using the EZNA Cycle Pure Kit from
Bio-Tek; The DNA concentrations of the purified PCR products were
quantified using NanoDrop.
Jaecklein 11
Transformation Amplification
The purified PCR product was transformed into One Shot TOP10 chemically competent
Escherichia coli cells using pCR4-TOPO vector from Thermo Fisher Scientific according to the kit
protocol (ThermoFisher Scientific; ). The
cloning reaction can be incubated for 5 to 30 minutes at room temperature. A 25-minute
incubation was used to ensure transformation. The transformed cells were spread over LuriaBertani (LB) plates treated with ampicillin antibiotic and 40 μL of X-Gal for screening and
incubated overnight at 37°C. The following day colonies were selected for colony PCR. Using
M13F and gene specific forward, as specified by the TOPO protocol, colony PCR was perform
using the following reaction conditions: 98°C for 5 minutes (1x); 95°C for 30 seconds, 58°C for
30 seconds, 72°C for 30 seconds (35x); 72°C for 7 minutes. All colonies selected for PCR were
grown on a master LB plate. Gel electrophoresis was performed to select for colonies with the
proper insert size using a 1% agarose gel at 120V for 45 minutes. Referring to the master plate,
bacterial colonies with the confirmed inserts were placed in liquid LB with ampicillin at 1:1000
ratio and left to culture overnight in 37°C with shaking. After overnight incubation, Plasmid Mini
bio-tek; After Plasmid MiniPrep, the DNA concentrations were
measured using NanoDrop.
Restriction Digestion
A digestion was performed using EcoRI and 600ng-1000ng of the purified MiniPrep DNA
for additional insert confirmation. EcoRI-HF from New England Biotech was used for the 35s
CaMV/ adh1 intron samples. The samples were digested for 1 hour at 37°C and run on a 1%
Jaecklein 12
agarose gel at 140V for 30 minutes. Tgs5 samples were run with EcoRI from Promega and
digested for 1, 4, and 6 hours at 37°C. Samples with confirmed insert size were then sent for
DNA Methylation Analysis
M13F primers were used for sequencing. The sequencing results were analyzed using
Kismeth plant bisulfite analysis program (Gruntman et al. 2008). Reference sequences were
taken from the Maize Genetics and Genomics Database, MaizeGDB, searching under B73
version-3 reference genome. Kismeth evaluates CG, CHG, and methylation, where H is an A,T
or C. Unmethylated cytosine residues are converted into uracil residues which will ultimately be
read as a thymine the final sequence. Any cytosine in the reference sequence that is matched
with a thymine in the bisulfite sequence is marked as unmethylated. Methylated cytosine
residues are left untouched by the bisulfite reaction; therefore, any cytosine in the reference
sequence that is matched with another cytosine in the sulfite sequence is marked as
Jaecklein 13
Control: 35sCaMV Promoter/ adh1 intron
Using the sequencing data, Kismeth revealed the expected methylation pattern in the
35S CaMV Promoter/ adh1 intron region.
Wild type tgr1 plants showed dense cytosine
methylation in the 35S CaMV promoter region and a lack of cytosine methylation the adh1
intron, just as expected from the previous description (Madzima et al. 2011). Mutant tgr1
plants showed a lack of cytosine methylation through the 35S CaMV promoter region and the
adh1 intron. The adh1 intron remained hypomethylated in wild type replicates. The 35S CaMV
promoter region in wild type tgr1 plants displayed an increase in cytosine methylation
compared to mutant individuals (Figure 3). Biological replicates could not be obtained for one
tgr1 mutant, P3, or one tgr1 wild type, G1. Three biological replicates were sequenced for both
wild types G2 and G3 and mutant P2. Only two biological replicates were successfully
sequenced for mutant P1. Ideally, every plant would be represented by 10 biological replicates.
Due the low number of biological replicates, this data serves as a more qualitative evaluation
rather than quantitative. The control indicates that the conversion was semi-successful, more
biological replicates are needed to draw more definitive conclusions.
Despite the low number of control replicates, Tgs5 analysis commenced. Following the
methods proposed above, sequencing data was obtained for Tgs5. Initial sequencing attempts
were prevented due to low similarity between the sequencing results and the reference
sequence. Using Clustal Omega from The European Bioinformatics Institute, a consensus
Jaecklein 14
sequence was generated from the sequencing data. This consensus sequence was compared to
the reference sequence and again exhibited extremely low similarity, indicating that the DNA
segment amplified using the TOPO procedure was not Tgs5. No methylation data could be
collected from the sequences.
Preliminary results from a previous methylation analysis showed good conversation in
one mutant tgr1 sample, with no methylation in the 35S CaMV promoter or in the adh1 intron.
Using DNA from this successful sample, Tgs5 was amplified, sequenced and methylation data
was obtained (Figure 4). Analysis showed low CHH methylation compared to CG and CHG in
tgr1 mutants. The results; however, are solely qualitative due to a low number of replicates and
lack of wild type data for comparison.
Transcriptional silencing of exogenous DNA is essential to proper development and
genome stability.
In plants, the RNA-dependent DNA methylation pathway (RdDM), an
epigenetic mechanism of transcriptional silencing, mediates transgene, transposable element,
and viral DNA silencing. The RdDM pathway has been shown to regulate the expression of
several endogenous genes in Arabidopsis thaliana (Gu et al. 2011; Vermeersch et al. 2013;
Zheng et al. 2010). Similar analysis of endogenous RdDM targets has not been conducted in Zea
mays, despite its invaluable role as a major agricultural crop or as a model organism for
fundamental genetics research, such as the discovery of transposable elements, or “jumping
genes” (McClintock 1950). This investigation was designed to elucidate RdDM involvement in
Jaecklein 15
endogenous gene regulation in maize. Unfortunately, the data generated was inconclusive and
did not provide an accurate representation of the intended target, Tgs5.
PCR, gel electrophoresis, and fast digestion were used to verify insert size in the
biological replicates. Despite size verification, sequencing data revealed low similarity to the
Tgs5 reference sequence. Additional analysis using Clustal Omega verified that the sequencing
data did not represent the promoter region of Tgs5, thus making it impossible to complete
methylation analysis for Tgs5. Re-evaluation of the experimental components revealed that the
inconclusive sequencing data is most likely a result of DNA damage and non-specific primer
binding. Degenerate primers allow for base shifting in the primary sequence, which is necessary
for bisulfite conversion. This flexibility decreases the specificity of the primer pairs.
Furthermore, bisulfite conversion alters the DNA sequence and subsequently diminishes
genome stability. The experimental procedure outlined above will provide adequate analysis of
RdDM involvement in regulation of the Tgs5 gene. It was successful in amplifying Tgs5 in a
previous attempt with this protocol. In order to improve sequencing results, new degenerate
primers with more specific primer binding should be designed. Vector transformation time
should be increased to the maximum (30 minutes) to ensure proper transfer. Evaluating more
clones using colony PCR and increasing gel run time (from 45 minutes to an hour) should
provide more biological replicates, improve ladder resolution, and increase size determination
efficacy. Additionally, experimentation was initially delayed because mutant tgr1 plants were
late to express the transgene and develop the purple phenotype. This delay in coloration was
mistaken for improper seed selection and the young plants were discarded. After two
unsuccessful plantings, seeds from a different individual with the same genetic background
Jaecklein 16
were planted and allowed to grow for three and a half weeks. Mutant plants were not
identifiable until two and a half weeks, much later than is observed in outdoor field conditions.
Once the purple phenotype presented, most of the plants were discarded leaving a collection of
ten healthy, purple tgr1 mutant plants and ten healthy, tgr1 green plants. The reason for the
delay is unclear; however, inconsistent artificial sunlight in the greenhouse is a potential causal
Operating under the assumption that the original hypothesis is true, RdDM regulates
the endogenous gene, Tgs5, it is expected that Kismeth results would reveal a hypomethylated
pattern in the promoter region of Tgs5 in tgr1 mutant plants compared to wild type individuals.
More specifically a decrease in CHH methylation (where H represents an A, T, or C) is expected,
because the Tgs5 gene is located near transposable elements in a gene rich region. Methylation
in gene rich regions is highly correlated with CHH methylation “islands” located upstream of
gene transcription start sites (TSSs), especially in genes in close proximity to transposable
elements (Gent et al. 2013; Qing Li et al. 2015; Q. Li et al. 2015; Lu et al. 2015). If the hypothesis
were false, Kismeth analysis would reveal no significant difference in methylation between
mutant and wild type individuals, which would indicate that RdDM and cytosine methylation
are not responsible for regulating this gene. The observed increase in Tgs5 expression in
mutants necessitates the involvement of regulatory machinery; however, no difference in
methylation between wild types and mutants would indicate that RdDM and cytosine
methylation are not involved in regulating this target.
However, the Tgs genes all demonstrated characteristics indicative of RdDM
involvement such as; close proximity to transposable elements, changes in expression levels
Jaecklein 17
and number of homologous 24-nt siRNAs in mutants tgr1 and mop1 individuals (Madzima et al.
Areas surrounding transposable elements demonstrate a decrease in expression
associated with methylation, which is indicative of RdDM mediated silencing (Qing Li et al.
2015; Q. Li et al. 2015; Lu et al. 2015). The Tgs genes demonstrated a twofold change in 24-nt
siRNA within 2kb of the TSS (Madzima et al. 2014). Additionally, these genes demonstrated a
change in expression in RdDM mutants. DNA methylation in the promoter regions and
transcription start sites directly link methylation to gene expression levels (Wang et al. 2015).
The involvement of 24-nt siRNAs paired with a change in gene expression also suggests that the
RdDM pathway regulates these Tgs genes.
Tgs5, in particular, was up regulated in the RdDM mutants, mop1 and tgr1 (Madzima et
al. 2014). Genes regulated by RdDM are expected to exhibit an increase in expression in RdDM
mutants due to loss of silencing. Rmr6 is an allele of mop3 and tgr1; it also codes for the largest
subunit of RNA polymerase IV (Hollick et al. 2005). Consequently, Rmr6 is also essential for
maintaining methylation at various points in the maize genome and influences gene expression.
Transcript analysis has confirmed up regulation of the Tgs5 gene in Rmr6 mutants, which
bolsters the hypothesis that RdDM regulates this endogenous gene in maize (Erhard et al.
2015). Several lines of evidence indicate RdDM in regulating Tgs5, making it the ideal target to
study RdDM regulation of endogenous genes in maize.
In maize, genome-wide methylation analysis of various tissues indicates that the
relationship between methylation and gene expression is not as simple as previously
interpreted (Gent et al. 2013; Qing Li et al. 2015; Q. Li et al. 2015; Lu et al. 2015; Wang et al.
2015). Although methylation of the 35S CaMV promoter is correlated with repression of the b1
Jaecklein 18
transgene, DNA methylation does not necessitate transcriptional gene silencing. The location
and specific variants of methylation, CG, CHG, and CHH all influence gene regulation in a unique
manner. For instance, a highly methylated CG region accompanied by regions of low CHG and
CHH methylation is implicated in enhancing gene expression (Lu et al. 2015; West et al. 2014).
Regardless of methylation’s effect, the epigenetic regulation constructed by RdDM pathway is
essential to plant health and development. Little is known about RdDM pathway’s role in
regulating endogenous genes, which was the purpose of this study. Unfortunately, sequencing
results did not adequately represent the endogenous target gene, Tgs5. In spite of the negative
results of this study, the Tgs genes, more specifically Tgs5, represents the most promising
target for studying RdDM regulation of endogenous genes. Previous research has shown that
Tgs5 gene expression is altered in RdDM mutants and it exhibits the hallmark characteristic of
RdDM targets such as proximity to transposable elements and changes in 24-nt siRNAs (Erhard
et al. 2015; Madzima et al. 2014). Future efforts to elucidate RdDM involvement in endogenous
gene regulation will focus on resolving experimental issues so the potential of Tgs5 and other
Tgs gene can be properly exploited.
I would like to thank Dr. Karen McGinnis and Dr. Thelma Madzima for their patience and
guidance throughout this project. I also thank Ji Huang and Linda Stroud, PhD candidates in the
McGinnis lab, for their lab mentorship.
Jaecklein 19
Figure 1: The b1 genomic transgene and associated phenotypes. (A) The transgene
includes the 35S cauliflower mosaic virus (35S CaMV) promoter and the alcohol
dehydrogenase 1 (adh1) intron to boost B1 expression. (B) The green (BTG-silent)
plants on the left represent the wild type tgr1 phenotype, green individuals are
either homozygous dominant or heterozygous. The purple (BTG-active) plants on
the right are homozygous recessive tgr1 mutants.
Figure 2: Seeds from a self-crossed heterozygous tgr1 plant were used. (A) Punnett
square representation of the expected genotypic and phenotypic outcomes based on
a heterozygous self-cross. Green (BTG-silent) and purple (BTG-active) individuals are
expected to appear in a 3:1 ratio. (B) A Chi-square analysis was performed based on
expected and observed values for the 60 seeds planted (57 out of 60 survived).
Analysis showed no significant difference between the observed and expected values
(df=1) with a Chi-squared value of 0.29.
Jaecklein 20
Figure 3: Bisulfite conversion methylation analysis of the control 35S CaMV Promoter region
and adh1 intron in tgr1 in two wild type (G1 and G2) and in two mutant (P1 and P2)
individuals. (A) Comparison of percent methylation of CG, CHG, CHH and total methylation in
wild tpe and mutant individuals shows an overall decrease in methylation in mutants. The
most dramatic decrease in methylation in mutants is observed in CHH methylation (B)
Kismeth methylation dot plot representation of the 35S CaMV promoter and adh1 intron
region. Green, blue and red coloration corresponds to CHH, CHG, and CG methylation,
respectively. Shaded circles represent methylated cytosine and unfilled circles represent
unmethylated cytosine. Focus on variability between wild type and mutant methylation in
the regions below the thick black bars.
Jaecklein 21
Figure 4: Graphical representation of preliminary results for Tgs5 methylation analysis in a
single tgr1 mutant sample. Complete bisulfite conversion was confirmed for this sample
using the 35S CaMV promoter/ adh1 intron control analysis. Only three biological replicates
were used for this data. It can be noted that in these particular samples this region of Tgs5
exhibited lower CHH methylation than CG or CHG methylation.
Jaecklein 22
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