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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 By: 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 Abstract: 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 Introduction: 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 Methods: 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 instructions (Zymo Research; https://www.zymoresearch.com). The converted DNA 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 amplification. 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 KM64 5’ AAAGGAYAGTAGAAAAGGAAGGTGG 3’ KM882 5’ CCCAAAATTACTCAACRAATCATAAACCAA 3’ Tgs5 KM1338 5’ TAGAGGATGTGYTTAAATGAYTTATAGTTTGG 3’ KM1339 5’ AAAAAAAATRAAAACACTARATRACAACCCAC 3’ 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 Omega Bio-Tek according to the manufacturer’s instructions (Omega Bio-Tek; http://www.omegabiotek.com). 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; https://corporate.thermofisher.com/en/home.html ). 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 Kit was executed according to the manufacturer’s protocol (Omega bio-tek; http://www.omegabiotek.com). 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 sequencing. 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 methylated. Jaecklein 13 Results: 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. Tgs5 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. Discussion: 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 factor. 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. 2014). 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. Acknowledgments 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 A B 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. A + - + +/+ +/- - +/- -/- B Mutant NullHypothesis BTG-Silent Plants BTG-Active Plants 2 tgr1 3:1 41 16 0.29 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 A MUT WT B 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 References Arteaga-Vazquez, M. A., & Chandler, V. L. (2010). Paramutation in maize: RNA mediated transgenerational gene silencing. (1879-0380 (Electronic)). Baucom, R. S., Estill, J. C., Chaparro, C., Upshaw, N., Jogi, A., Deragon, J. M., . . . Bennetzen, J. L. (2009). Exceptional Diversity, Non-Random Distribution, and Rapid Evolution of Retroelements in the B73 Maize Genome. PLoS Genetics, 5(11), e1000732-e1000732. doi: 10.1371/journal.pgen.1000732 Chandler, V. L. (2010). Paramutation's Properties and Puzzles. Science, 330(6004), 628-629. 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