Faculty of Science AN EXPLORATORY ANALYSIS OF GENE EXPRESSION IN DWARF MISTLETOE (ARCEUTHOBIUM AMERICANUM) USING qRT-PCR 2017 | DAKOTA JONES B.Sc. Honours thesis - Biology An Exploratory Analysis of Gene Expression in Dwarf Mistletoe (Arceuthobium americanum) Using qRT-PCR by Dakota Jones A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF BACHELOR OF SCIENCE (HONS.) in the DEPARTMENT OF BIOLOGICAL SCIENCES (C.M.M.B.) This thesis has been accepted as conforming to the required standards by: Joanna Urban (M.Sc.), Thesis Supervisor, Dept. Biological Sciences Cynthia Ross Friedman (Ph.D.), Co-supervisor, Dept. Biological Sciences Naowarat Cheeptham (Ph.D.), Examining Committee member, Dept. Biological Sciences Dated this 5th day of May, 2017, in Kamloops, British Columbia, Canada © Dakota Jones, 2017 i Abstract: Dwarf Mistletoe (Arceuthobium americanum) exhibits some very interesting characteristics, such as being a hemi-parasitic plant-on-plant parasite. It’s most interesting and most characteristic feature is its seed dispersal via explosive discharge. This seeds dispersal method is very reminiscent of a cannon shooting a cannonball. The mechanism of how the small female fruit are able to do this is currently unknown with different theories trying to explain it. The theory with the most backing is a thermogenesis theory. As the fruit is monumentally smaller than the trees it infects it has a theoretical infinite source of water to draw from. Coupled with the fruits ability to trap water with a waxy viscin and produce heat via thermogenesis it is thought that the fruit will use water pressure to propel its seed. As water is drawn in, heat is generated, expanding the water and creating pressure as the water is trapped by the wax until it ruptures the cuticle and expels the seed. This is the basis of this study as it aims to investigate the genetic side of this theory. A previous study using the Affymetrix microarray heterologous probe approach revealed the potential presence and expression of key developmental genes. Four of these key genes for this theory were studied: 14-demethylase, Alkaline Invertase, Cer1, and Shine 3. Each of these genes was run through quantitative real-time polymerase chain reaction with primers designed upon plant various genetic libraries (tobacco, wine grapes, maize, Arabidopsis, etc.). Various cDNA sample extracted from dwarf mistletoe were also ran. These samples were extracted over the growing season to the time of seed dispersal from mature (3rd year) female fruits. RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) shown to be constitutively expressed and was used as reference gene. By comparing the Cq values over the growing season of each gene a picture of the genetic expression patterns of the genes could be seen. 14-demethylase was shown to have Cq values of June = 38.07, July = 35.69, and August = 36.28. This shows the genes expression increasing from June to July where it peaks and falls off in August. The Alkaline Invertase showed a similar pattern of expression with June = 30.32, July = 26.83, and August = 31.47. The Cer1 and Shine3 genes showed inconclusive results after several runs and many adjustments to the procedure. From the data collected, it may be possible that that the 14-demethylase and Neutral Invertase are preparative, helping with protection and energy storage prior to dispersal respectively. To solidify any speculations around these results further research into the genetics of this plant needs to be conducted. ii Thesis supervisors: Cynthia Ross Friedman, and Joanna Urban iii Acknowledgements: This project involved the help of many different people without whom this project would not have been possible. First I would like to thank my supervisors Joanna Urban and Cynthia Ross Friedman. They were both always able to help me with any question I have had as well as providing feedback every step of the way and being just one email away. I would also like to thank Jon Van Hamme, who helped me troubleshoot different issues I had using the qRT-PCR. Tim Bientjes and Dylan Ziegler also helped me by doing necessary PCR runs and helping me with plant physiology aspects respectively. Lastly, I want to thank the UREAP committee for selecting me as a recipient of the UREAP scholarship which funded this project and allowed me to pursue this project. iv Table of Contents Abstract......................................................................................................................................... ii Acknowledgements.......................................................................................................................iii List of Figures................................................................................................................................vi List of Tables.................................................................................................................................vi Introduction....................................................................................................................................1 Background......................................................................................................................................1 Neutral Invertase..............................................................................................................................2 14-Demethylase...............................................................................................................................2 Cer1.................................................................................................................................................3 Shine3..............................................................................................................................................3 Methods..........................................................................................................................................4 Collection and Preparation..............................................................................................................4 Primer Design.................................................................................................................................4 qRT-PCR Setup...............................................................................................................................4 Results............................................................................................................................................8 Neutral Invertase.............................................................................................................................8 14-Demethylase.............................................................................................................................11 Cer1 and Shine3.............................................................................................................................15 Discussion.....................................................................................................................................18 Presence of Genes..........................................................................................................................18 Neutral Invertase...........................................................................................................................18 14-Demethylase.............................................................................................................................15 Cer1 and Shine3.............................................................................................................................20 Future Work.................................................................................................................................21 Conclusion....................................................................................................................................21 Literature Cited..........................................................................................................................23 Appendix .....................................................................................................................................26 v List of Figures Figure 1. Example of plate layout for qRT-PCR ...........................................................................8 Figure 2. Neutral Invertase Amplification Plot............................................................................. 9 Figure 3. Neutral Invertase Cq value graph..................................................................................10 Figure 4. Neutral Invertase Melting Curve...................................................................................11 Figure 5. 14-demethylase Amplification Plot...............................................................................12 Figure 6. 14-demethylase Cq Value graph...................................................................................13 Figure 7. 14-demethylase Melting Curve.....................................................................................14 Figure 8. Cer1 Amplification .......................................................................................................15 Figure 9. Cer1 Melting Curve.......................................................................................................16 Figure 10. Shine3 Amplification Plot...........................................................................................17 Figure 11. Shine3 Melting Curve..................................................................................................18 List of Tables Table 1. qRT-PCR mixture used for RuBisCO...............................................................................5 Table 2. qRT-PCR mixture used for Neutral Invertase...................................................................6 Table 3. qRT-PCR mixture used for 14-demethylase.....................................................................6 Table 4. qRT-PCR mixture used for Cer1......................................................................................6 Table 5. qRT-PCR mixture used for Shine3...................................................................................7 Table 6. Parameters used for qRT-PCR..........................................................................................7 Appendix Table 1A. Primer data...................................................................................................................26 Table 2A. qRT-PCR run raw data.................................................................................................26 vi Introduction Background Dwarf Mistletoe (Arceuthobium americanum) is a very interesting little plant that inhabits the North-Western America, mainly the British Columbian and Albertan regions of Canada1. In this region it will infect its primary host of lodgepole pines (Pinus Contorta subsp. latifolia) as it is a hemi-parasitic organism2. Being hemi-parasitic, Dwarf Mistletoe is able to get nutrients from its host, but is still able to provide other necessities for itself by using photosynthesis. The parasitic aspect of the plant causes vascular damage to the host as the plant is able to tap into the tree’s vascular system, decreasing the quality of the wood1. Once infected, the tree’s branches will proliferate and bundle cause what is known as “brooming”. This causes vast economic losses in the lumber industry as the wood becomes knotty, resinous, and unusable. Controlling the spread of this parasite has become an important aspect of research into this organism, not only for scientists but also the lumber industry. The parasite takes numerous years until it is fully mature and able to disperse it seeds to its next host3. It takes 7 years for the plant to complete its life cycle, from the time of infection to the time it flowers. At the end of the 5th year, the male will pollinate the female and will take another year for the females to develop their fruits and then disperse them. The fruits are dispersed using a method known as “explosive discharge”. As the name suggests the seed explodes from the fruit and is propelled to a new host. This process generates an immense amount of pressure within the fruit without a clear reason as to how it achieves it4. The leading hypothesis as to how this occurs is that thermogenesis triggers the dispersal of the seeds4,7. As the fruit matures it will produce vesicular cells2 capable of producing hydrophobic lipids5,7. At the same time it is drawing in more nutrients and water7, which will be trapped in the hydrophobic coating of the lipids6,7. The ability to undergo thermogenesis means that the fruit could draw in water to heat up within the lipid coating4. As liquid is heated it will expand, so as the water expands it will push against the lipids and build up pressure, to a point where it eventually ruptures through the fruit and is expelled to its next host. 1 The thermogenic hypothesis is the basis of this study as the genetic aspects of the theory was being examined. Previous microarray assays7 had revealed a possible presence of developmental genes, key to the theory7, which opened the door for genetic investigation as well as further testing to support the findings. Four genes were selected for testing: Neutral Invertase, 14-demethylase, Cer1, and Shine3. Each of these genes would be investigated through qRT-PCR to measure the expression levels of each of the genes from the time of fruit development to the time of dispersal, which is roughly, May to September of the fruits 6th year. Neutral Invertase This enzyme is only found in the cytosol or bound to organelles of plants and certain photosynthetic bacteria, responsible for the conversion of sucrose to glucose and fructose8. Sucrose is the main molecule used to store energy collected by photosynthesis, but needs to be broken into a useable source, glucose, making its conversion imperative in many organisms9. With this in mind this unique sucrose cleaving enzyme an essential in many plants10. More than being able to breakdown sucrose, neutral invertase has shown the ability to control the amount of sucrose that a cell will uptake11. This essentially means that this enzyme controls the amount of energy the cell gets as it by mediating the transport of sucrose into the cell. As dwarf mistletoe is hemi-parasite on a plant as well as being able to perform photosynthesis2, it is logical to assume it will have an abundance of sucrose as it energy storage source. This will lead to the need for a sucrose cleaving enzyme, such as neutral invertase. As the fruit has an increased need for nutrients as it matures, the uptake of sucrose will increase greatly6. Neutral invertase would be able to allow more sucrose in as well as converting it to useable glucose8. An increase in the expressed Neutral Invertase gene as the fruit matures to the time of seed dispersal is expected. 14-demethylase 14-demethylase is a highly conserved and essential enzyme seen in every kingdom of life12. This enzyme is a critical step in the production of cholesterol in animals and bacteria, ergosterols in fungi, and sterols in plants13. The production of sterols, mainly sitosterols, is 2 crucial to maintaining the fluidity of the plasma membrane in plants. As thermogenesis occurs in dwarf mistletoe the plasma membrane will become stressed4, jeopardizing the integrity of the membrane. With the production of more sterols during thermogenesis, the membrane will be protected12,13. In addition these sterols are highly hydrophobic and will act to trap the water inside of the fruit13. Cer1 To further trap water in its fruit dwarf mistletoe secretes lipids from its vesicular cells2, some of which are waxes. Waxes are highly hydrophobic and commonly found on the epicuticular layer of plants, to serve as a barrier to harm, as well as cuticular or subcuticular for seed protection14. These waxes have very long-chain fatty acids attached to them, making them extremely hydrophobic, keeping water from passing through it. Cer1 has been identified as an initiator of wax biosynthesis in many plants. It has also exhibited signs of responding to stress factors such as temperature15. These characteristics make it a good fit for the thermogenetic theory, as it can trap the water and respond to the high heat of thermogenesis. Shine3 Similar to Cer1, Shine3 is involved in the biosynthesis of waxes in plants, helping the retention of water in plants as well as helping serve as a defense layer16,17. This gene may have the ability to respond to other stress factors, such as water loss, allowing for up-regulation when water is lost16. An attribute such as this could allow for more water retention in the Dwarf Mistletoe as it approaches the time of seed dispersal, as to insure enough water to generate explosive pressure. Another key aspect of this gene is that it has illustrated functions in stomata density17. Stomatal density and explosive discharge have been shown to have a correlation, as the density decreases over the growing season of mature females to the time of seed dispersal18. This is important as stomata help control temperature, with a decrease in density leading to an increase in temperature. This would aid in heating the fruit, thus generating more pressure for dispersal. 3 Methods and Materials Collection and Preparation Female dwarf mistletoe samples were collected from a previously used site of infected lodgepole pines. The research site was near Stake Lake, just south of Kamloops, British Columbia, Canada (50° 31′ latitude and 120° 28′ longitude). The samples were taken 2 weeks from late May to late August for the years 2012-2016. Once collected the female fruits were flash frozen using liquid nitrogen and crushed into a fine powder to be used for mRNA extraction. mRNA was extracted using an Epicentre Master Pure RNA Extraction Kit to be used for reverse transcription to obtain cDNA. Reverse transcription was achieved by using a Applied Biosystems High Capacity cDNA Reverse Transcription Kit, at a concentration of approximately 30ng and working solutions diluted to 3ng. Primer Design All primers were design using a combination of plant genomic libraries and microarray assay results7. The plant genomic libraries consisted of many different plants, such as tobacco, maize, wine grapes, cocoa, Arabidopsis, etc. For the Affymetrix microarray assay, the heterologus probes allowed for some sequence data to be seen. As they are small probes and don’t have to be exact matches, the exact sequence of the genes can’t be determined solely by this method. Once all the libraries were compiled, they were overlaid to find highly conserved regions or conserved start and stop codons. In the case of the microarray assay, areas that exhibited high fluorescence were selected as the base for the primers. This is due to the probes used in microarray assays, which fluoresce more greatly with the amount of bonds it makes with the test strain. Based on this, the probe with high amounts of fluorescence should be the closest match to the gene it is targeting. All primers were 25-30bp with a melting temp of 58-60°C, with products ranging from 100-300bp, as well as having no internal dimerization. This was performed using a combination of NCBI BLAST© and the Northwestern University OligoCalc©. Upon completion and delivery of the primers, each was run through a base PCR run to test for possible amplification. If the primers would show signs of amplification, then they would be ran through qRT-PCR, while ones that did not work, were no longer used. Each primer was run 4 using a concentration gradient to determine what ratio of primer to mastermix would give the best results. qRT-PCR Setup The basic outline for the setup was determined by a previous study using qRT-PCR to find a constitutively expressed gene in dwarf mistletoe19. Previous findings have established RuBisCO as having equal expression levels throughout the growth period. RuBisCO is a major component of photosynthesis, as it acts to fixate CO2, producing energy for the dwarf mistletoe20. This allows RuBisCO to be used as a reference reading for the qRT-PCR runs, as it will be expressed at the same level over the growth period. Runs from the same month but from different years were used to keep yearly variances from affecting results. Each gene that was to be run through the qRT-PCR needed to be a mixed with all other necessary components required to run. The mixes used for the genes in this experiment are as follows: Table 1. Mixtures used for running RuBisCO through qRT-PCR Single Batch Triplicate Batch cDNA 1µL 3µL Forward Primer (91) 0.75µL 2.25µL Reverse Primer (92) 0.75µL 2.25µL Mastermix SYBR® Green 5µL 1µL H2O 2.5µL 7.5µL Total 10µL 30µL 5 Table 2. Mixtures used for running Neutral Invertase through qRT-PCR Single Batch Triplicate Batch cDNA 1µL 3µL Forward Primer (93) 1µL 3µL Reverse Primer (94) 1µL 3µL Mastermix SYBR® Green 5µL 1µL H2O 2µL 6µL Total 10µL 30µL Table 3. Mixtures used for running 14-demethylase through qRT-PCR Single Batch Triplicate Batch cDNA 1µL 3µL Forward Primer (89) 1µL 3µL Reverse Primer (90) 1µL 3µL Mastermix SYBR® Green 5µL 1µL H2O 2µL 6µL Total 10µL 30µL Table 4. Mixtures used for running Cer1 through qRT-PCR Single Batch Triplicate Batch cDNA 1µL 3µL Forward Primer 0.75µL 2.25µL Reverse Primer 0.75µL 2.25µL Mastermix SYBR® Green 5µL 1µL H2O 2.5µL 7.5µL Total 10µL 30µL 6 Table 5. Mixtures used for running Shine3 through qRT-PCR Single Batch Triplicate Batch cDNA 1µL 3µL Forward Primer 1µL 3µL Reverse Primer 1µL 3µL Mastermix SYBR® Green 5µL 1µL H2O 2µL 6µL Total 10µL 30µL Each experiment was run using the same parameters, based on the melting point of the primers designed and previous studies19. This was done as it produced the best results and was another way to standardize each run. The following depicts the parameters used for each experiment: Table 6. Parameters used for qRT-PCR runs Uracil-DNA Temperature Time Cycles 50°C 2 minutes 1 95°C 10 minutes 1 95°C 10 seconds 60°C 30 seconds 95°C 15 seconds 15°C 15 seconds 95°C 15 seconds Glycolase Incubation Polymerase activation PCR Melt Curve 40 1 7 Figure 1. An example of the plate layout to be used in a qRT-PCR run (U= Unknown/experimental, N= Non-template control) Results Neutral Invertase Using primers 93 and 94 (See Appendix), the target gene, Neutral Invertase, was amplified and an expression pattern of the gene over the growth period was obtained (Figure 1). 8 Neutral Invertase July Neutral Invertase August Neutral Invertase June RuBisCO (Green) Figure 2. Amplification plot of a qRT-PCR run using Neutral invertase primers with cDNA from June, July, and August of 2012. (June mean Cq=31.32, July Mean Cq = 26.84, August Mean Cq =33.22, RuBisCO Mean Cq = 35.89 ) From overlaying the results of the runs, the Cq values of neutral invertase over the growing season are (n=6) (Figure 2.), with RuBisCO averaging at 37.76±1.39. This shows that the expression of this gene increase from June to July, where it plateaus, and drops slightly in August. 9 32 31 Cq Value 30 Neutral Invertase 29 28 27 26 25 24 June July Month August Figure 3. A graph of the mean Cq values (generated by EcoStudy software) for Alkaline Invertase over the growing season The melting point of each gene was tested against its non-template control, as it makes sure the primers were binding to the target gene and not dimerizing (Figure 3.). 10 Experimental Non-template Control Figure 4. The melt curve of Neutral Invertase, showing the 2 peaks of the experimental mixture and the non-template control. Data collected from the melt curve show 2 different peaks for the non-template control and experimental runs. This suggests that the primers were able to target Neutral Invertase in the presence of cDNA. Even though the peaks are at very similar temperatures, the fluorescence exhibited by both differs greatly. These difference point to the possibility of the primer-dimer and primer hybrid the having the same melting temperature, but dimerization occurs rarely. 14-demethylase With the use of primers 89 and 90 (See Appendix), an expression pattern for 14demethylase over the growing season was obtained (Figure 4.). 11 14-demethylase July 14-demethlyase August 14-demethylase June RuBisCO (Green) Figure 5. Amplification plot of a qRT-PCR run using 14-demethylase primers with cDNA from June, July, and August of 2012. (June mean Cq=N/A, July Mean Cq = 35.73, August Mean Cq = 36.28, RuBisCO Mean Cq = 37.20) By looking at the all the data collected for the 14-demethylase runs, it seems that the expression pattern follows that of Neutral Invertase. The expression increases from June (Cq=38.07±0.53) to July (Cq = 35.69±0.52), and then dropping slightly in August (Cq= 36.28±0.90) (Figure 5.). RuBisCO overall mean Cq was 37.76±1.39. 12 39 38.5 38 37.5 14-demethtylase Cq Values 37 36.5 36 35.5 35 34.5 34 33.5 June July Month August Figure 6. A graph of the mean Cq values (generated by EcoStudy software) for 14demethylase over the growing season Melting curve data was also collected for the 14-demethylase runs, which produced Figure 5. 13 Experimental Non-template Control Figure 7. The melt curve showing the separate peaks of the 14-demethylase experimental and non-template control runs. The differences in peaks between the experimental and non-template control runs are a bit hard to see, as the control is covered by the experimental. Once you are able to see the difference in the peaks though it becomes clear that the peaks are at very different temperatures, nearly 5°C apart. This means that the products of the experimental and non-template control had very different products. This supports the idea that primers were able to bind their target sequence in the presence of cDNA, but dimerized when there was no cDNA. 14 Cer1 and Shine3 Despite numerous runs with different primers and adjusted procedures, no useable data for Cer1 or Shine3, was collected. As these genes are not very well studied there was a lack of sequenced genes to base the primers off of. This is a possible reason as to why there were no usable results, as the primers were not specific enough and were not able to bind to their target genes. Figure 8. Amplification plot of a qRT-PCR run using Cer1 primers with cDNA from June, July, and August of 2012. No amplification was achieved 15 Figure 9. The melt curve of Cer1, there is no way to discern any different peaks. 16 Figure 10. Amplification plot of a qRT-PCR run using Cer1 primers with cDNA from June, July, and August of 2012. No amplification was achieved 17 Figure 11. The melt curve of Shine3, there is no way to discern any different peaks. Discussion Presence of Genes With the runs using the primers targeting Neutral Invertase and 14-demethylase, it is not fully certain, but it seems the microarray assay results are supported. Due to the primers design, using conserved regions of known genetic sequences in other plants, it points to these genes are also present in dwarf mistletoe. While we are not able to definitively confirm their existence with these results, these results strongly suggest they do. Neutral Invertase As Neutral Invertase is a sucrose cleaving enzyme, also able to control sucrose intake8, it was suspected that the expression of this gene would increase, dropping the mean Cq, as the fruit approaches seed dispersal. The results produced by this experiment suggested otherwise, as the 18 expression of Neutral Invertase increase from June to July and dropping slightly into August, the time of seed dispersal. A previous study suggested, they same result, as the measured expression levels of Neutral Invertase dropped into September19. The Affymetrix results also should similar results as the expression of this gene was down regulated in September, when compared to May7.These results would suggest that Neutral Invertase acts preparative, rather than directly in the seed dispersal. This is still highly speculative but it is possible that Neutral Invertase is able to store the glucose in a vacuole to be used at the time of thermogenesis. While sucrose is an excellent bulk storage molecule, it needs to be converted into a usable form, glucose, to be used in most organisms8. At the time of thermogenesis, the dwarf mistletoe fruit will need an immense amount of energy to heat its water, thus building pressure4. The demand for glucose, and subsequently sucrose would be enormous, which creates problems. Sucrose cannot freely diffuse the plasma membrane into the cell; they need to be taken in using a form of active transport using a biochemical gradient or energy8. If sucrose is actively transported by using ATP, then during thermogenesis taking in the required amount of sucrose would require a large amount of energy, taking away from what is needed for thermogenesis. This is a very inefficient way to get the energy needed for discharge. If sucrose was acquired using a biochemical gradient, it is likely that it would slow down greatly, or even halt, during thermogenesis. As the sucrose is taken into the cell the concentrations on either side of the membrane would level out, ceasing the force driving sucrose across23. Even if the concentration gradient did not collapse, Neutral Invertase can only operate at a certain rate and probably be unable to supply enough glucose needed for thermogenesis. Other invertases, mainly the Acidic Invertase, have been shown to be sugar converting enzymes, able to store sugars in vacuoles21. With glucose being readily available to the fruit, a steady stream of glucose can be fed to the mitochondria to perform thermogenesis. At the same time Neutral Invertase could be converting more sucrose at a lesser rate to meet the energy demand. Another possible benefit of the plant doing this is that glucose will affect the tonicity greater, encouraging water uptake23. As sucrose is a disaccharide, it will be split into glucose and fructose, two monosaccharides. Having two sugars will make the fruit more hypertonic, than 19 with a single sugar. This will cause the fruit to take in more water to dilute the sugars, which will be trapped in the wax and used for discharge. 14-demethylase Similarly to the Neutral Invertase expression, it was expected that 14demethylase would have an increased expression over the growth period, peaking in August. It was found that the expression pattern of 14-demethylase was the same as Neutral Invertase, increasing from June to July, where it peaks, and dropping slightly into August. The previous Affymetrix results exhibited similar results, as the expression rate of 14-demethylase decreased in September when compared to the rates in May7. As 14-demethylase is crucial for the production of sterols which will maintain the integrity of the plasma membrane during thermogenesis12, it was expected that it would be expressed the most near to the time of discharge. As 14-demethylase is expressed the most in July, about a month before discharge, preemptive expression of this gene would have a benefit being preparative. This is pure speculation as no definitive conclusions can be drawn with the current information available. With the ability to keep membrane fluidity and retain water in plants12, it would make sense that producing sterols prior to discharge would be more beneficial than producing them around the time of discharge. If a mass of sterols was made before there was any membrane stress it would serve to be more protective than producing at the time of stress. When thermogenesis is triggers it will not cease or halt, it will proceed continuously until the seed is discharged4. This being the case, producing sterols to combat this at the time of discharge would mitigate a small amount of damage. While if the sterols were produced in bulk prior and implemented into the plasma membrane, it would negate a greater amount of damage, as compared to producing simultaneously. Another possible reason for a spike in expression prior to discharge has to do more with the ability of sterols to be precursor hormones13. Sterols can be the backbone of many different signaling hormones, such as brassinosteroids in plants, making them critical in many signal pathways. It is possible that that spike in 14-demethylase expression could be to produce more signaling molecules to trigger a specific pathway, if not multiple pathways. As to what the 20 pathways would active is unknown. It could be used for development, protection13, anatomy changes18, or any other signalling pathway. Cer1 and Shine3 Cer1 and Shine3 results came back inconclusive, with no usable data, with no clear reason as to why it did not work. The most likely reason for these results is that the primers were not able to bind their target sequence. Both of these genes are highly unstudied or sequenced, with the majority of the data around these genes is in Arabidopsis. This makes it difficult to build any primers that would work for dwarf mistletoe, as there could be a vast difference in the sequence between the organisms. Even the Affymetrix results are based on Arabidopsis, as the probes are based on Arabidopsis genes. To be able to design more specific primers, capable of targeting these genes more genetic work into these genes in dwarf mistletoe or more similar organisms will need to be done. This will build up sequence data that will more accurately portray dwarf mistletoe. Future Work For Cer1 and Shine3 it is very apparent that a better genetic understanding of the genes is required. Without this understanding production of better primers, able to amplify their target genes will be prove to be challenging. Both of these genes are very important to the thermogenetic theory of discharge making further research into these genes is imperative. Neutral Invertase runs gave an insight into the expression pattern of this gene, and its possible function in the fruit. To definitively determine its function and possible glucose storage functions, an in vitro study on the accumulation or movement of glucose and sucrose would need to be conducted. As for 14-demethylase its function can be simple, helping support the plasma membrane, or complex, acting in signal pathways. To flush out its true function, a better understanding of dwarf mistletoe genetics needs to be obtained before any tests on this gene can be made. This is the limiting factor if dwarf mistletoe research, as this plant is not as widely studied as others, many internal factors and functions remain enigmatic. Due to the sensitive nature of this assay it is easy to cause errors in procedure, obscuring the results. Possible photo-bleaching, due to high light exposure may have occurred making the fluorescent ability of the primers to be compromised. 21 Conclusion The thermogenic theory of dwarf mistletoe’s seed dispersal, although not definitively proven, has become more strongly supported the more this plant is studied4. The results of this study seem to support this theory again, from a more genetic perspective. The increase of Neutral Invertase, prior to the explosive discharge, could possibly point to a way to draw in more water23 and store energy for use in thermogenesis for discharge21. 14-demethylase expression pattern data showed the possibility of building up a heat resistant, hydrophobic coating prior to discharge, negating temperature damage12. Also the 14-demethylase could serve as important component for biosynthesis of signal molecules, triggering a myriad of functional pathways13. While the exact process of these genes are known, they functionality in the seed dispersal of dwarf mistletoe is undeniable. Interpretation of this studies result seems to support the microarray assay results performed by Joanna Urban7. These results are not definitive as the Cq values were very high, often above 30, which means that there is very little RNA, making it hard for the results to carry much weight. While highly speculative, interpretation of the key developmental genes from this study may help understand the genetic profile of this organism. 22 Literature Cited 1. McIntosh, R.2004. Dwarf Mistletoe: Ecology and Management. Forest Service, Saskatchewan Environment. 2. Ross-Friedman, C., and Sumner, M. 2009. Maturation of the Embryo, Endosperm, and Fruit of the Dwarf Mistletoe Arceuthobium americanum. International Journal of Plant Science [Internet]. [cited 2017 April 6]; 170(3): 290-300. doi: 10.1086/596328 3. Brandt, JP.2006. 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List of successful primers with relevant information Creator Date Primer Sequence Organism Joanna Urban Joanna Urban Joanna Urban Apr15 Apr15 Apr15 GGTTATGGAAGCAGAGG Arceuthobium ATTAC oxycedri GGCTGGCTGTTAAGATG Arceuthobium ATTAG oxycedri AAACGGATAATGTCCCG Arceuthobium CAC oxycedri Joanna Urban Joanna Urban Joanna Urban Apr15 Apr15 Apr15 ACTTCGTCATCGTACACT Arceuthobium CGC oxycedri TTGTGGATCTTGACGGCT Arceuthobium GC oxycedri TCGTAATATTCCGGCCA Arceuthobium CGAG oxycedri Gene # Previous ID Sterols 89 Ao Sterols F Sterols 90 RUBISC O 91 Ao Sterols R Ao RUBiSCO F Ao RUBISCO R Ao Alk Inv F Ao Alk Inv R RUBISC O 92 Alk Invertase 93 Alk Invertase 94 Table 2A. Raw data for qRT-PCR runs Well A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 Sample Assay Name Name June '12 93/94 93/94 August '13 93/94 93/94 June '12 93/94 93/94 August '13 93/94 93/94 June '12 93/94 93/94 August '13 93/94 93/94 June RuBisCo Assay Role Cq Cq Mean Cq Std. Dev. Unknown 31.34125 31.29737 0.132624 NTC 30.49408 30.1689 2.30476 Unknown 32.97135 33.2151 NTC 32.97074 30.1689 0.760937 2.30476 Unknown 31.14837 31.29737 0.132624 NTC 30.78711 30.1689 2.30476 Unknown 32.6059 33.2151 NTC 32.37097 30.1689 0.760937 2.30476 Unknown 31.4025 31.29737 0.132624 NTC 31.49768 30.1689 2.30476 Unknown 34.06804 33.2151 0.760937 NTC 30.54253 30.1689 2.30476 Unknown 33.47309 35.19466 2.449056 26 '12 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 D1 D2 D3 D4 D5 D6 D7 D8 E1 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 D1 D2 D3 D4 D5 August '13 June '12 August '13 June '12 August '13 July '12 Aug '12 July '12 Aug '12 RuBisCo NTC 37.93898 1.39124 RuBisCo Unknown 36.5964 RuBisCo NTC 36.5964 N/A 37.93898 1.39124 RuBisCo Unknown 34.14619 35.19466 2.449056 RuBisCo NTC 37.93898 1.39124 RuBisCo Unknown RuBisCo NTC 36.5964 N/A 37.93898 1.39124 RuBisCo Unknown 34.33313 35.19466 2.449056 RuBisCo NTC 37.93898 1.39124 RuBisCo RuBisCo 93/94 93/94 93/94 93/94 93/94 93/94 93/94 93/94 93/94 July '12 Aug '12 June '12 89/90 89/90 July '12 89/90 89/90 Aug '12 89/90 89/90 June '12 89/90 89/90 July '12 89/90 89/90 Aug '12 89/90 89/90 June '12 89/90 89/90 July '12 89/90 Unknown NTC NTC Unknown Unknown NTC Unknown Unknown NTC Unknown Unknown 27.36845 25.79912 26.9779 25.7691 26.74717 26.00062 29.71941 27.98433 26.26415 36.5964 37.93898 30.1689 26.84354 26.41422 30.1689 26.84354 26.41422 30.1689 26.84354 26.41422 N/A 1.39124 2.30476 1.095787 0.505629 2.30476 1.095787 0.505629 2.30476 1.095787 0.505629 Unknown NTC Unknown NTC Unknown NTC 35.52222 35.59795 35.08488 36.45031 35.81014 34.61873 35.12511 35.71184 35.6876 35.71184 36.27574 35.71184 0.541396 0.984976 0.519097 0.984976 0.895121 0.984976 Unknown NTC Unknown NTC Unknown NTC 35.34471 34.68465 35.33811 35.18855 35.0938 34.53036 35.12511 35.71184 35.6876 35.71184 36.27574 35.71184 0.541396 0.984976 0.519097 0.984976 0.895121 0.984976 Unknown 34.50842 35.12511 0.541396 NTC 37.39876 35.71184 0.984976 Unknown 36.50726 35.6876 0.519097 27 D6 D7 D8 E1 E2 E3 E4 E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 C1 C2 C3 C4 C5 89/90 Aug '12 89/90 89/90 June '12 RuBisCo RuBisCo July '12 RuBisCo RuBisCo Aug '12 RuBisCo RuBisCo June '12 RuBisCo RuBisCo July '12 RuBisCo RuBisCo Aug '12 RuBisCo RuBisCo June '12 RuBisCo RuBisCo July '12 RuBisCo RuBisCo Aug '12 RuBisCo RuBisCo Jun '12 89/90 89/90 July '12 89/90 89/90 Aug '12 89/90 89/90 Jun '12 89/90 89/90 July '12 89/90 89/90 Aug '12 89/90 89/90 Jun '12 89/90 89/90 July '12 89/90 89/90 Aug '12 89/90 89/90 Jun '12 RuBisCo NTC 35.68638 35.71184 0.984976 Unknown 35.72231 36.27574 0.895121 NTC 35.99504 35.71184 0.984976 Unknown 38.82623 35.19466 2.449056 NTC 38.92273 37.93898 1.39124 Unknown 38.05794 36.61513 0.964789 NTC 37.93898 1.39124 Unknown 39.77469 N/A NTC 37.93898 1.39124 Unknown 35.19466 2.449056 NTC 37.93898 1.39124 Unknown 36.49936 36.61513 0.964789 NTC 37.93898 1.39124 Unknown 39.77469 N/A NTC 37.93898 1.39124 Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown 35.07035 36.0313 37.63008 36.46004 36.01695 36.05733 35.42451 34.83225 37.18724 35.00007 34.61109 35.73958 37.07671 37.3809 36.36759 35.19466 37.93898 36.61513 37.93898 39.77469 37.93898 N/A 35.71184 35.6876 35.71184 36.27574 35.71184 N/A 35.71184 35.6876 35.71184 36.27574 35.71184 N/A 35.71184 35.6876 35.71184 36.27574 35.71184 N/A 2.449056 1.39124 0.964789 1.39124 N/A 1.39124 0.984976 0.519097 0.984976 0.895121 0.984976 0.984976 0.519097 0.984976 0.895121 0.984976 0.984976 0.519097 0.984976 0.895121 0.984976 28 C6 C7 C8 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 E5 E6 RuBisCo July '12 RuBisCo RuBisCo Aug '12 RuBisCo RuBisCo Jun '12 RuBisCo RuBisCo July '12 RuBisCo RuBisCo Aug '12 RuBisCo RuBisCo Jun '12 RuBisCo RuBisCo July '12 RuBisCo RuBisCo Aug '12 RuBisCo RuBisCo NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC Unknown NTC 37.93898 36.74132 36.61513 37.93898 39.77469 37.93898 N/A 36.95522 37.93898 35.36112 36.61513 37.93898 39.77469 39.77469 37.93898 N/A 37.93898 36.4159 36.61513 37.93898 39.77469 37.93898 1.39124 0.964789 1.39124 N/A 1.39124 1.39124 0.964789 1.39124 N/A 1.39124 1.39124 0.964789 1.39124 N/A 1.39124 29