TOWSON UNIVERSITY OFFICE OF GRADUATE STUDIES BIOINFORMATICS TOOLS AND ANALYSIS TO IDENTIFY CANDIDATE LETHAL GENES IN ROOT-KNOT NEMATODE (RKN) AND ITS COMPARISON WITH SOYBEAN CYST NEMATODE (SCN) INFECTION IN SOYBEAN By Ahmed I. Ismail A dissertation in partial fulfillment of the requirement for the degree Doctor of Science in Information Technology. Department of Computer and Information Science Towson University Baltimore, MD 21252 August 2013 ii iii Acknowledgment I would never have been able to finish my dissertation without the guidance of my committee members, help from friends, and support from my family and wife. I would like to express my deepest gratitude to my advisor, Dr. Nadim Alkharouf, for his excellent guidance, caring, patience, and providing me with an excellent atmosphere for doing research. I would like to thank Dr. Benjamin F. Matthews, for his assistance, guidance, patiently corrected my writing, and for providing me with the microarray data to be analyzed in our experiment. Special thanks go to Dr. Hammel and Dr. Hong, who were willing to participate in my final defense committee and for their valuable discussions and accessibility. I would like to thank Omar Darwish, who as a good friend was always willing to help and give his best suggestions. It would have been a lonely lab without him. I would also like to thank my parents, younger brother, and younger sister. They were always supporting me during the long years of my education and encouraging me with their best wishes. Finally, I would like to thank my wife. She was always there cheering me up and stood by me through the good times and bad. iv Abstract BIOINFORMATICS TOOLS AND ANALYSIS TO IDENTIFY CANDIDATE LETHAL GENES IN ROOT KNOT NEMATODE (RKN) AND ITS COMPARISON WITH SOYBEAN CYST NEMATODE (SCN) INFECTION IN SOYBEAN. Ahmed I. Ismail Root-Knot nematode (RKN; Meloidogyne spp.) is one of the most devastating obligate parasites that infect the roots of thousands of plant species. In the United States, the cost of the losses that these parasites cause annually is estimated to be about $8 billion. The RKN genome sequence is not well characterized or fully sequenced. We sought to identify genes that are lethal at any stage of RKN development. These genes could act as targets for RNAi constructs or gene knockout experiments. To that end we compared RKN gene sequences to the well-characterized and fully sequenced genome of the free-living nematode C. elegans (Caenorhabditis elegans). The primary database for C. elegans (www.wormbase.org) contains a huge repository of RNAi and mutation experiments on C. elegans genes. We conducted a comparative genomics analysis, comparing RKN genes of six species: Meloidogyne arenaria, M. chitiwoodi, M. hapla, M. incognita, M. javanica, and M. paranaensis to C. elegans via WormBase. The RKN sequences were obtained from nematode.net. Our analysis yielded many candidate lethal genes between Meloidogyne spp. and C.elegans. Some of these genes have already been tested experimentally and indeed found to be lethal to RKN. We also created a relational web based database that links the RKN genes and proteins to their C. elegans homologs. The database provides v search capabilities for scientists in the field. The database was created using SQL- Server 2008. The web based user interface was created using ASP.NET. Using microarray analysis we examined the expression of soybean (Glycine max) genes in roots infected by the RKN, Meloidogyne incognita at 12 days and 10 weeks after infection and similarly soybean roots infected by SCN at 8 days after infection. Gene expression was monitored using the Affymetrix Soybean GeneChip containing 37,500 G. max probe sets. A comparative microarray analysis was done between RKN and SCN using bioinformatics tools such as MATLAB (R2010b) Bioinformatics toolbox and Blast2go. The goal was to identify the common genes that were differentially expressed (induced or suppressed) as a result of the soybean infection by RKN and SCN. Our results showed that there are major differences in the gene expression profile between the soybean infected by SCN and the soybean infected by RKN. Where only 135 genes were shown to be induced and 82 genes were shown to be suppressed as the common altered genes between soybean roots infected by RKN (Meloidogyne incognita) and similarly soybean roots infected by SCN. vi Table of Contents Contents Acknowledgment ........................................................................................................................... iii Abstract .......................................................................................................................................... iv Table of Contents .......................................................................................................................... vi List of Tables ............................................................................................................................... viii List of figures ................................................................................................................................. ix List of Abbreviations .................................................................................................................... xi Chapter 1 ........................................................................................................................................ 1 Specific Aims and Objectives ........................................................................................................ 1 Chapter 2 ........................................................................................................................................ 5 Identifying Lethal Genes in RKN ................................................................................................. 5 2.1 Background and Significance ................................................................................................ 5 2.1.1 Damage ........................................................................................................................... 6 2.1.2 Life Cycle........................................................................................................................ 7 2.1.3 ESTs and Genome of RKN ............................................................................................. 8 2.2 Methodology of development .............................................................................................. 10 2.2.1 ESTs and Genomic Analysis......................................................................................... 12 2.2.2 RNAi Phenotypes discovery ......................................................................................... 14 2.3 Results .................................................................................................................................. 15 2.3.1 Pairwise alignment ........................................................................................................ 15 2.3.2 Significance groups ....................................................................................................... 16 2.3.3 Lethal Stages ................................................................................................................. 18 2.4 Conclusion ........................................................................................................................... 19 Chapter 3 ...................................................................................................................................... 20 Microarray Data Analysis ........................................................................................................... 20 3.1 Introduction on Microarray .................................................................................................. 20 3.2 Introduction to SCN ............................................................................................................. 21 3.2.1 Damage ......................................................................................................................... 21 3.2.2 Life Cycle...................................................................................................................... 22 3.2.3 Soybean resistance to SCN ........................................................................................... 24 3.3 Experimental Design ............................................................................................................ 25 vii 3.4 Materials and Methods ......................................................................................................... 26 3.4.1 Plant and nematode materials ....................................................................................... 26 3.4.2 RKN microarray data processing .................................................................................. 28 3.4.3 SCN microarray data processing ................................................................................... 33 3.4.4 Annotation..................................................................................................................... 34 3.5 Microarray results ................................................................................................................ 35 3.6 Discussion ............................................................................................................................ 63 3.7 Conclusion ..........................................................................................................................