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Gulsen2004a.Pdf Buffalograsses: Their Organelle DNA, Chinch Bug Resistance Variation, and Peroxidase Enzyme Responses to Chinch Bug Injury By Osman Gulsen A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Agronomy Under the Supervision of Professors Robert (Bob) C. Shearman and Kenneth P. Vogel Lincoln, Nebraska December, 2004 Buffalograsses: Their Organelle DNA, Chinch Bug Resistance Variation, and Peroxidase Enzyme Responses to Chinch Bug Injury Osman Gulsen, Ph.D. University of Nebraska, 2004 Advisors: Robert (Bob) C. Shearman and Kenneth P. Vogel Information on genetic diversity and relationship of native buffalograss germplasm is limited and genetic basis of agronomic traits is unknown. The objectives of this research were to determine: 1) the genetic diversity, relationships, and organelle DNA inheritance based on cpDNA and mtDNA, 2) chinch bug resistance variation in natural buffalograss populations characterized for cpDNA and mtDNA; 3) the degree of correlation between total protein content, basal peroxidase level, chinch bug injury, and ploidy level, and 4) total protein content and peroxidase changes of resistant and susceptible germplasm in response to chinch bugs. Fifty-six, 48,28, and, 6 buffalograsses were evaluated for organelle DNA, chinch bug resistance, correlation analysis, and peroxidase changes, respectively. Six cpDNA and three mtDNA non-coding regions were amplified by polymerase chain reaction, using universal chloroplast and mitochondrial primer pairs. Each amplified fragment was digested with 2 to 6 different restriction enzymes. For the chinch bug study, genotypes were evaluated in replicated trials under greenhouse conditions. Leaf samples were collected for peroxidase changes from infested and control plants at 7, 14,21, and 28 day after exposure (DAB) to chinch bugs. Peroxidase analyses were carried out using native gels stained for anionic peroxidases and enzyme kinetics were measured with a spectrophotometer. Forty-seven of 56 genotypes had identical cpDNA and mtDNA RFLPs and the rest showed only a few polymorphic markers, which suggests a single maternal origin for the four buffalograss ploidy levels. Based on the use of cpDNA primers amplifying intergenic region between psbC and tmS genes, and restriction enzyme Rae III, cpDNA was determined to be maternally inherited in buffalograss. The germplasm had considerable diversity for chinch bug resistance, with approximately 10% of the germplasm having a high resistance level. There was no significant correlation between chinch bug resistance and ploidy level or chinch bug resistance and pubescence. Of the genotypes studied, 4 were highly resistant, 22 were moderately resistant, 19 were moderately susceptible and three were highly susceptible to chinch bug injury, showing a continuous distribution. Basal peroxidase expression levels measured in the 28 non-infested plants of resistant and susceptible buffalograsses did not correlate with chinch bug injury. All six genoptypes evaluated for chinch bug activity showed an increased level of peroxidase levels in infested plants, suggesting upregulation in response to chinch bug injury. Relatively low levels of peroxidase in a highly chinch bug resistant genotype, PX-3-5-1, infers contribution of other genes to chinch bug resistance. Overall results indicate substantial genetic variation in buffalograss germplasm that can be used to enhance buffalograss breeding programs and increase understanding of the chinch bug resistance mechanism. To mother Huriye, my bellowed wife Fatma, and dear sons Askin and Kerem, and daughter Sumeyra ACKNOWLEDGEMENTS First and foremost I would like to express my thanks and gratitude to Dr. Robert C. Shearman and Dr. Kenneth P. Vogel. Under their guide I have been encouraged to explore my potential in plant science. I am especially grateful for their guidance during the apparently endless pursuit of this degree. I would also like to thank the other members of my guidance committee, Drs. Tiffany M. Heng-Moss, Donald J. Lee, and P. Stephen Baenziger, who provided inspiration and a friendly environment at UNL. I believe that these are the key issues in career development. Their collaboration greatly helped me to accomplish my expectations at UNL. I remember that my mother encouraged me to get higher education all the time and for her endless support. I am very thankful and will remember her support forever. My wife, Fatma Funda, deserves special consideration here. Her constant support in all stages of this thesis has been an important part of the pursuit of my PhD degree. I truly appreciate her love, patience, loyalty and assistance. My special thanks are to members of buffalograss research group for sharing ideas and setting up my experiments at the Department of Entomology: Terrance P. Riordan, Frederick P. Baxendale, Thomas E. Eickhoff, Hikmet Budak, Wyatt G. Anderson, and others. Carol Caha, Ismail Dweikat, and Herbert Siqueira backed me up all the times when needed. I also thank to the staff personnel at the Department of Agronomy and Horticulture, and Entomology. Finally, I thank the Department of Agronomy and Horticulture of University of Nebraska, Lincoln for the financial support for my PhD program here and for giving me opportunities to discover new research areas and myself, and Ministry of Agriculture of Turkey for permitting me PhD study here. Finally I thank Allah for giving me chance to accomplish this program, and hope to use my knowledge I gained here for humanity. TABLE OF CONTENTS Page List of Tables iii List of Figures .iv Literature Review 1 Chapter 1. Organelle DNA Diversity among Buffalograsses From The Great Plains Of North America Using CpDNA and MtDNA RFLPs Abstract 49 Introduction 51 Materials and Methods 54 Results and Discussion 57 Literature Cited 62 Chapter 2. Buffalograss Germplasm Resistance to Blissus occiduus Hemiptera: Lygaeidae) Abstract. 75 Introduction 76 Materials and Methods 78 Results and Discussion 81 Literature Cited 86 Chapter 3. Total Protein and Peroxidase Enzyme Changes Among Chinch Bug Resistance and Susceptible Buffalograsses Abstract 92 Introduction 94 ii Materials and Methods 97 Results and Discussion 103 Literature Cited 108 Appendix 120 11l LIST OF TABLES Table Page 1.1 Buffalograss genotypes and their ploidy levels, sex expression and geographic origins used in organelle DNA study 69 1. 2 Primer pairs, corresponding regions, and reannealing temperatures used in organelle DNA study '" 72 1.3 Chloroplast and mitochondrial DNA primer pairs, restriction enzymes, and number of restriction fragments scored in each digestion 73 1.4 Pairs of cpDNA and mtDNA primers and PCR product sizes used in studies of Buchloe, Citrus, and Quercus 74 2.1 Susceptibility of buffalograss genotypes to Blissus occiduus under greenhouse conditions, and their ploidy level, mean chinch bug numbers and pubescence 89 3.1 Buffalograss genotypes used in chinch bug evaluation study, their ploidy levels, and chinch bug resistance 113 Al CpDNA and mtDNA RFLP raw data file 120 A2 Correlations among chinch bug injury, ploidy levels, total protein content, and basal peroxidase level.. 125 A3 Analysis of variance for plant total protein content. 126 A4 Analysis of variance for peroxidase specific activity changes 127 iv LIST OF FIGURES Figure Page 1.1 UPGMA dendogram of 56 buffalograsses and two outgroups 66 1.2 CpDNA restriction fragments of buffalograss accessions and two outgroups 67 1.3 Verification of maternal inheritance of cpDNA in buffalograsses 68 2.1 Distribution of chinch bug resistance among buffalograss genotypes 88 3.1 Total protein contents of infested and non-infested plants of six genotypes .115 3.2 Changes in total protein contents among the six infested and non-infested genotypes 116 3.3 Changes in peroxidase specific activity through 7, 14,21, and 28 DAE among the six genotypes .117 3.4 Native gels stained for anionic peroxidase activity 14 DAE 118 3.5 Native gels stained for anionic peroxidase activity 28 DAE 119 Al Neighbor-Joining tree based on organelle DNA RFLPs and produced by Mega software 128 A2 Minimum evolution tree based on organelle DNA RFLPs and produced by Mega software '" 129 A3 UPGMA tree based on organelle DNA RFLPs and produced by Mega software .130 A4 Native gel analysis of infested and non-infested plants of six v genotypes 7 DAB to chinch bugs 131 A5 Native gel analysis of infested and non-infested plants of six genotypes 21 DAB to chinch bugs 132 A6 Polymorphism for anionic peroxidases among 28 buffalograsses 133 1 LITERATURE REVIEW Introduction Buffalograss [Buchloe dactyloides (Nutt.) Engelm.] is a stoloniferous, perennial warm season grass species that is native to the North American Great Plains (Wenger, 1943). It performs well under warmer temperatures. High temperatures tend to increase photosynthetic capacity (Monson et aI., 1983). Although buffalograss performs acceptably under low rainfall and relative humidity, it shows rapid growth when grown under irrigated conditions (Beetle, 1950; Riordan, 1991). Turf type buffalo grasses have been used in home lawns, school grounds, parks, roadsides, cemeteries, golf course fairways and roughs due to the drought resistance and low maintenance requirement (Savage and Jacobson, 1935; Beard, 1973; Riordan, 1991; Fry, 1995; McCarty, 1995). Its aggressive stoloniferous growth habit and dense sod forming capabilities make it an excellent conservation species (Wenger,
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