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Characterization of Fraxinus Spp. Phloem Transcriptome Thesis

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Characterization of Fraxinus Spp. Phloem Transcriptome Thesis Characterization of Fraxinus spp. Phloem Transcriptome Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Loren J Rivera Vega, B.S. Graduate Program in Entomology The Ohio State University 2011 Thesis Committee Omprakash Mittapalli, Advisor Pierluigi E Bonello Daniel A Herms Abstract Ash trees (Fraxinus spp.) are widely spread throughout eastern North America and represent an important tree species in urban landscape and natural settings. Since the accidental introduction of the invasive insect pest emerald ash borer (Agrilus planipennis Fairmaire) millions of ash trees have been killed by this devastating pest. North American species such as black (F. nigra), white (F. americana), green (F. pennsylvanica), and to some extent blue (F. quadrangulata) are susceptible to this pest. However, in Asia, A. planipennis’ natural habitat, the damage to native ash is isolated and only observed in trees under stressed conditions, indicating some level of resistance, presumably due to a shared co-evolutionary history. To date various efforts to contain A. planipennis have been implemented, yet it continues to spread at an alarming rate within the US and Canada. Despite the high impact status of A. planipennis, there is little information available at the molecular level for any Fraxinus species. The main objective of this study was to characterize the transcriptome of ash phloem including North American and Manchurian species, which would then lay the foundation for future functional and applied studies. The first part of the study was to describe the phloem transcriptome and predict potential molecular markers using 454 pyrosequencing. A database of more than 50,000 sequences was obtained from a pooled sample of black, green, white, blue and Manchurian ash. The database was profiled using ii Blast2GO software in order to annotate the sequences, determine gene ontology (GO), and identify associated metabolic pathways. Also, the expression of eight candidate genes was quantified using real time quantitative PCR (RTqPCR) in three ash species: black, green and Manchurian ash. Finally, more than 1,272 single nucleotide polymorphisms (SNPs) and 980 microsatellites were predicted. The information obtained from the first part of the study was used to carry out the second part of the study, which was profiling of housekeeping genes across various ash species. In total, ten housekeeping genes were analyzed in order to identify a reliable reference gene for use in gene expression techniques such as RTqPCR. This analysis identified translation elongation factor alpha (eEF1α) as the most stable gene, and therefore is recommended for current and future ash transcriptomics work. The third and final part of the study was to compare and contrast constitutive gene expression profiles of black, green and Manchurian ash using RNA-Seq on an Illumina platform. From this study, three more databases were generated for ash, one for each of the three species analyzed. These databases were also profiled using Gene Ontology terms and KEGG pathways, results were similar to those observed in the first part of the study. More than 50,000 SNPs and 5,000 microsatellites were predicted from all three databases combined. Differential analysis revealed approximately 600 genes to be differentially expressed among the ash species out of the 8,691 orthologs used for the analysis. In order to validate the results obtained from these gene expression profiles, eight candidate genes were analyzed using RTqPCR. iii The data generated from these studies using next-generation sequencing strategies has revealed a wealth of genomic information on ash, the target host for A. planipennis. The results obtained will clearly lay the foundation for future functional studies, ash breeding programs, candidate gene identification, and population genetic studies, as well as simply provide more (comparative) genomic information for deciduous trees. iv Dedication To my friends and family: Those far away who keep me grounded and those close who keep me going. v Acknowledgements I would like to thank: My advisory committee members Dr Omprakash Mittapalli for his constant motivation, Dr Daniel Herms for all his advices including those that didn‟t involve research and Dr Enrico Bonello. Ronald Batallas, Lucia Orantes, Alejandra Claure, and Nelson Davila, for those great first days as interns during which we shared our hopes and dreams – truly the end of an era. The Mittapalli Lab: Priya Rajarapu, Ma Anita Bautista, Binny Bhandary and Praveen Mamidala for their support both inside and outside of the laboratory. The Herms Lab: Bryant Chambers, Diane Hartzler, Vanessa Muilenburg, Priya Loess, and Jamie Imhoff for welcoming me into their lab and always considering me a part of it. AGEAP-OSU for their support and guidance, for showing me that despite being far away from my country as long as a Zamorano is around we‟ll always feel at home. Los padrinos – the Cañas-Acosta family – for opening the doors of their home and allowing me to share with them so many special moments, for their invaluable advices, and mainly for their friendship. vi All my friends in Ohio for allowing me to be my crazy self and still love me and for letting me be a part of their lives. All my friends in Honduras/Latin America because I was always able to feel their love and support despite the distance and time. My amazing family for always supporting and trusting my decisions. SEEDs Grant Program and USDA-APHIS for the funding provided for this research. vii Vita 2007……………………………………… B.S. Agricultural Science and Production Pan-American College of Agriculture Tegucigalpa, Honduras 2008-2009……………………………….. Research Aide Department of Entomology The Ohio State University, Wooster, OH 2009-present………………………………Graduate Research and Teaching Assistant The Ohio State University, Wooster, OH Publications Rivera-Vega L*, Mamidala P*, Koch JL, Mason ME, Mittapalli O. 2011. Evaluation of reference genes for expression studies in ash (Fraxinus spp). Plant Molecular Biology Reporter. DOI 10.1007/s11105-011- 0340-3. Bai X*, Rivera-Vega L*, Mamidala P, Bonello P, Herms DA, Mittapalli O. 2011. Transcriptomic signatures of ash (Fraxinus spp) phloem. PLoS ONE 6(1): e16368. Mittapalli O, Rivera-Vega L, Bhandary B, Bautista M, Mamidala P, Michel A, Shukle R, Mian R. 2011. Cloning and characterization of mariner-like elements in the soybean aphid, Aphis glycines Matsumura. Bulletin of Entomological Research 12:1-8 Rivera-Vega L and Mittapalli O. 2010. Molecular characterization of mariner–like elements in emerald ash borer, Agrilus planipennis (Coleoptera, Polyphaga). Archives of Insect Biochemistry & Physiology. 74(4): 205-216. Bhandary B, Rajarapu SP, Rivera-Vega L, Mittapalli O. 2010. Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR. Journal of Visualized Experiments (39). Saballos A, Vermerris W, Rivera L and Ejeta G. 2008. Allelic Association, Chemical Characterization, and Saccharification Properties of brown midrib Mutants of Sorghum (Sorghum bicolor (L.) Moench). Bioenergy Research. 1: 193-204 Field of Study Major Field: Entomology viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgements ............................................................................................................ vi Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... ix List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiv Chapter 1 ............................................................................................................................. 1 Introduction ..................................................................................................................... 1 1.1 Framework ............................................................................................................. 1 1.2 Research objectives ............................................................................................... 7 1.3 References ............................................................................................................. 8 Chapter 2 ........................................................................................................................... 15 Transcriptomic Signatures of Ash (Fraxinus spp.) Phloem .......................................... 15 2.1 Abstract ................................................................................................................ 15 2.2 Introduction ......................................................................................................... 16 2.3 Materials and Methods ........................................................................................ 18 2.3.1 Sample Collection and RNA extraction .......................................................
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