Introduction

1. PCR Analysis as a Possible Means for Sex Determination of Eastern Redcedar (Juniperus virginiana) Jaclyn Knibbe, Dr. Elizabeth Heeg-Truesdell, Dr. Todd Tracy Biology Department, Northwestern College, Orange City Iowa Female Red Cedar Male Red Cedar Abstract – Although native to northern U.S., the eastern red cedar (Juniperus virginiana) has quickly become invasive in areas of the Midwest where natural regeneration of planted trees is occurring. Because of the negative impact of red cedar on pastureland and prairies, ecologists have begun finding ways to control its spread. The best known methods include controlled burns and cutting the trunk at its base. However, adult eastern red cedars, once burned, can still cast large shadows on nearby plants needing sunlight.  Additionally, the dense, prickly foliage of the tree make it difficult to cut the trunk. We propose an alternative method via sex determination of seedlings by means of PCR analysis.  That is, female eastern red cedars could be identified via PCR and either not planted or removed as seedlings in a more efficient manner than if they were allowed to grow to maturity.  Farmers would still benefit from the eastern red cedar’s maintenance-free windbreaks, but without the hassle of their eventual invasion into pastureland and prairie.  Our research investigated a gene (Accession: AF151429) that codes for a pollen protein, and we tested whether female red cedars lack this gene and can thus be identified and removed before maturity. Introduction The invasive nature of the eastern red cedar has become a significant problem with farmers in the Midwest and surrounding areas. The current methods for mass removal of the juniper involve controlled burns or cutting the tree at the base of its trunk. Controlled burns, however, leave larger adult trees still standing. These large and dense foliaged adults can still cast great shade upon the surrounding prairie life. Cutting the tree at its base can become a difficult procedure as well. The foliage is very thick and spiky and requires thick gloves to prevent cuts. The eastern red cedar is a rare plant species (among 4%) in that it has separate male and female individuals. If we can find a gene that is only found in one sex, we can select against it. In Papaya plants, for instance, once the gene for a male was found, they amplified that gene with PCR and noted that only males contained a band in the gel electrophoresis. Those seedlings containing the male band could then be removed. Similarly, our research began by finding the sex-specific gene located in the eastern red cedar. A pollen gene was found with nucleotide BLAST on PubMed The gene was then examined if it was a sex-specific gene or not. (Figure 2) Figure 2: PCR steps 1) Denaturation: Heat briefly to separate DNA strands 2) Annealing: cool to allow primers to form hydrogen bonds with ends of target sequence. 3) Extension: DNA polymerase adds nucleotides to the 3’ end of each primer. Figure 1: The red cedar was originally concentrated in Wisconsin and northern Minnesota. Fire suppression allowed for the red cedar to spread through the Great Plains and eastern. Discussion After making changes to the protocol and changing PCR temperatures and duration time periods, we were able to extract enough DNA for a successful PCR reaction. (Figure 4) This gave bands in the 500bp region, which indicated that the pollen gene (520bp) was located and copied. Female 2 did not show a band, however. Because primer-dimers did show up, it was determined that the solution was at least loaded. Two conclusions could then be made. Either no DNA was extracted from female 2 (considering the difficulty in DNA extraction with conifers), or females 1 and 3 were false positives. Contamination could have occurred where male DNA got into the female DNA. With proper lab techniques used, however, this possibility was unlikely. Gloves were used throughout the entire procedure and filter tips were used for transferring solutions. To make a definite conclusion, a positive control was needed. Photosystem II primers from the BioRad (2008) primer kit were used as a positive control (Figure 5). Photosystem II is the first protein complex in light-dependent photosynthesis. Every plant that photosynthesizes contains the gene for photosystem II at a length of 455bp. The eastern red cedar photosynthesizes, and therefore contains the gene for photosystem II. Figure 5 showed that both males and females contained a band around 500bp. This concluded that the photosystem II primers found DNA and were able to make copies in the PCR reaction. Since female 2 did not show a band for photosystem II, it must have not contained any extracted DNA to begin with. Therefore, females 1 and 3 were not false positives and contained the pollen gene. We conclude then, that female Juniperus virginiana contains the gene sequence for pollen formation. This is not entirely uncommon, however. Most organisms that separate into male and female during development contain most of the same genetic information. It is the transcription factors (normally located on the Y chromosome in males) that make the physical, expressed differences between males and females (Gilbert 2003). In the case of the eastern red cedar, females have the pollen gene but lack the expression factors necessary to produce pollen protein. Conversely, eastern red cedar females contain expression factors for berry production while males do not. Figure 2: Gene sequence of the pollen gene. For better PCR accuracy, only half of the gene was framed by primers (circled in red). The band length around 520 bp should also show up after gel electrophoresis to confirm the success of the primers. Red Cedar Benefits *Dense foliage makes great windbreaks *Low maintenance; grows quickly *Heartwood is good for furniture and for warp-sensitive musical instruments. Red Cedar Disadvantages *Reproduces quickly *Shades out and steals nutrients from native prairie life *Has become highly invasive in pasturelands Figure 3: Graphical representation of hypothesized PCR product bands. If females don’t contain the pollen gene, they will not contain a PCR product band. Results: • Figure 4: • Bands show up around 500 bp, indicating the pollen gene (520bp) was successfully detected by the primers and copied. Female 2 does not contain a band giving two possible conclusions: • Females 1 and 3 are false positives for the pollen gene • Because DNA extraction was not always 100% successful, Female 2 did not contain any extracted DNA initially. Pollen Gene The pollen gene (accession: AF151429) was initially located to find differences between several allergenic conifers. Messenger RNA (mRNA) from pollen protein was taken and converted into cDNA in order to determine its sequence (Figure 2). Because the pollen gene was converted back to cDNA from mRNA, it is possible to use the sequence for seedlings. If the gene sequence was pure mRNA, seedlings would not likely be producing pollen and therefore would not contain mRNA. It was hypothesized that only male red cedars would contain the pollen gene and would only contain a band from the PCR product. Since females wouldn’t contain the gene, the primers would not locate the specific sequence and would not make copies. (Figure 3) Because two conclusions were a possible from the result of the gel from Figure 4, another PCR reaction was performed with a positive control using photosystem II primers. Photosystem II is the first protein complex in light-dependent photosynthesis. Every plant that photosynthesizes (including the eastern red cedar) contains the gene for photosystem II at a length of 455bp. Therefore, both male and female plants should contain a band for photosystem II if DNA is present. Future Directions: Since the pollen gene is not located on the Y-chromosome, it can not be used as a sex-determinate gene. However, a northern blot could be performed to screen for the presence of mRNA. Since only males produce pollen, they would only be producing mRNA to make the pollen protein. Similarly, a western blot could be performed to screen for the presence of the pollen protein. Yet, the goal of our research is to determine the sex of an individual when it has not yet matured. Seedlings would not likely produce pollen until they reached reproductive maturity. Therefore, it might be difficult to find any pollen mRNA or protein in seedlings. A second option is to find the transcription factors that initiate the expression of the pollen gene. Females contain the pollen gene, but since pollen is not produced, they must lack the expression factors needed. This would require more research in that these expression factors have not yet been found and decoded. Only a select number of genes have been decoded for the eastern red cedar. If transcription factors become too difficult to locate, we could start from scratch and locate a completely new gene hypothesized to be on the Y-chromosome. From past research, the genes located on the Y-chromosome have been found in dioecious plants. Research by Ling, I. et al. (2003) indentified a sex-associated gene in the tree, Ginkgo Biloba. “Of the 8,372 RAPD bands, only a 682 bp RAPD maker generated by a primer (S1478) of random decamer squence, named S1478-682, was found to be associated with the male plants” (Ling 2003). To find a specific RAPD marker takes a lot of research filled with many trials and errors. We can search in the same region that the Ginkgo sex-specific marker is to find a possible homolog for the eastern red cedar sex-determinate gene. Once again, this will require further decoding of Juniperus virginiana genes. As more research is done on the eastern red cedar, more decoded genes will be available for sex-determination. In the meantime, the best way to remove invasive cedars remains with controlled burns and cutting at the trunk. Methods The DNA extraction protocol was adapted from C.S. Kim et al., (Oxford Journal, 1997). Red cedar samples were collected using the lid of a 1.5 ml microcentrifuge tube to clip off pieces of the needles, which prevented any contamination. Needles were ground up with a mortar and pestle in one drop of 1% 2-mercaptoethanol and 300 [mu]l of extraction buffer (250 mM NaCl, 25 mM EDTA, 0.5% SDS, 200 mM Tris-HCl pH 8.0). After incubating at room temperature for one hour, freshly prepared PVP (10,000 MW PVP, Sigma, 6% of final volume) and ½ volume of 7.5 M ammonium acetate were added individually. The solution was incubated on ice for 30 minutes and centrifuged for 10 minutes (10,000 g). The supernatant was then transferred to a new tube and 1 vol isopropanol is added and then incubated at -20º C overnight. The mixture was centrifuged at 10,000 g and the supernatant was discarded. The DNA pellet was dried by blotting on a paper towel. DNA pellet was resuspended in 500 [mu]l of TE buffer (10 mM Tris-HCl ph 8.0, 0.1 mM EDTA pH 8.0). The mixture was placed in a boiling water bath for 5 minutes. One vol chloroform-isoamyl alcohol (24:1) was added and emulsified by inverted shaking. Another vol of chloroform-isoamyl alcohol (24:1) was added and emulsified. Once centrifuged (10,000 g) for 5 minutes, the supernatant was transferred to a new tube and 1 vol of isopropanol was added and incubated at -20º C for 10 minutes. After centrifuging (10,000 g) for 10 minutes, the pellet was dried by blotting on a paper towel and then resuspended in 30 [mu]l of TE buffer. Best PCR procedure for primers: Denature: 94°C….2 minutes Loop (x40): Denature 94°C….1 minute : Anneal 59°C…….1 minute : Extend 72°C…....2 minutes Final Extension: 72°C…….10 minutes Hold 4°C Figure 5: The positive control, photosystem II, shows that Female 2 did not contain successfully extracted DNA. Pollen gene bands show to be around 500bp and photosystem II bands show up around 500bp as well. This also concludes that both male and female eastern red cedars may contain the gene for pollen. References: Brennan, S., and Withgott, J. (2005). Environment: the science behind the stories. Pearson/Benjamin-Cummings: San Francisco. Farjon, A. (2005). Monograph of Cupressaceae and Sciadopitys. Royal Botanic Gardens, Kew. Ferguson, Edwin R. and Edwin R. Lawson. (1974). Eastern Redcedar…an American Wood. Czarapata, Elizabeth. (2005). Invasive Plants of the Upper Midwest. University of Wisconsin Press: Madison. De Jong, Thomas J. and Peter G.L. Klinkhamer. (2005). Evolutionary Ecology of Plant Reproductive Strategies. Cambridge University Press: Cambridge. Davis, Mark A. (2008) Invasive Species: Kudzu. Available from: <http://www.biologyreference.com/Ho-La/Invasive-Species.html> [Accessed 7 December 2008] Forseth, Irwin N. Jr and Anne F. Innis. (2004). Kudzu: History, Physiology, and Ecology Combine to Make a Major Ecosystem Threat. Critical Reviews in Plant Sciences: Vol. 23 Issue 5. p401-413. Global Invasive Species Database (GISD), (2005a).  Welcome to the global invasive species database. Available from: <http://www.issg.org/database> [Accessed 8 December 2008] Lockwood, J., Hoopes, M., and Marchetti, M. (2007). Invasion ecology. Blackwell Publishing: Malden. EOL; Encyclopedia of Life. (2008). Juniper. Available from: <http://www.eol.org/taxa/16914138> [Accessed 7 December, 2008] Tracy, Todd. (2007). Invasive Species and the Call to Christian Environmental Stewardship. Department of Biology, Northwestern College: Orange City. Acknowledgements: Funding for this project was provided by a Northwestern College Scholarship Grant and a grant from the Iowa Science Foundation.