Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2005 Genomic markers for the study of Ostrinia nubilalis population dynamics and traits conferring resistance to Bacillus thuringiensis (Bt) crystalline toxins Brad Steven Coates Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Entomology Commons, and the Genetics Commons Recommended Citation Coates, Brad Steven, "Genomic markers for the study of Ostrinia nubilalis population dynamics and traits conferring resistance to Bacillus thuringiensis (Bt) crystalline toxins " (2005). Retrospective Theses and Dissertations. 1855. https://lib.dr.iastate.edu/rtd/1855 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Genomic markers for the study of Ostrinia nubilalis population dynamics and traits conferring resistance to Bacillus thuringiensis (Bt) crystalline toxins by Brad Steven Coates A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Daniel F. Voytas, Co-major Professor Leslie C. Lewis, Co-major Professor Richard L. Hellmich Fredric J. Janzen John D. Nason Jonathan F. Wendel Iowa State University Ames, Iowa 2005 Copyright© Brad Steven Coates, 2005. All rights reserved. UMI Number: 3184610 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3184610 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Brad Steven Coates has met the thesis program requirements of Iowa State University Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. For the Majbr Program Ill TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES vi ABSTRACT vii CHAPTER 1. GENERAL INTRODUCTION 1 Introduction 1 Dissertation Organization 2 Literature Review 3 References 31 CHAPTER 2. PARTIAL MITOCHONDRIAL GENOME SEQUENCES OF OSTRINIA NUBILALIS AND OSTRINIA FURNACALIS Abstract 48 Introduction 48 Materials and Methods 49 Results and Discussion 51 References 55 CHAPTER 3. GEOGRAPHIC AND VOLTINISM DIFFERENTIATION AMONG NORTH AMERICAN OSTRINIA NUBILALIS (EUROPEAN CORN BORER) MITOCHONDRIAL CYTOCHROME C OXIDASE HAPLOTYPES Abstract 60 Introduction 60 Materials and Methods 62 Results and Discussion 66 References 73 CHAPTER 4. POLYMORPHIC CA/GT AND GA/CT MICROSATELLITE LOCI FOR OSTRINIA NUBILALIS (LEPIDOPTERA:CRAMBIDAE) Abstract 83 Short Note 83 References 87 CHAPTER 5. TWO SEX-CHROMOSOME-LINKED MICROSATELLITE LOCI SHOW GEOGRAPHIC VARIANCE AMONG NORTH AMERICAN Abstract 89 Introduction 90 Materials and Methods 91 IV Results 94 Discussion 95 References 99 CHAPTER 6. SEQUENCE VARIATION IN THE CADHERIN GENE OF OSTRINIA NUBILALIS'. A TOOL FOR FIELD MONITORING Abstract 105 Introduction 105 Materials and Methods 108 Results and Discussion 112 References 119 CHAPTER 7. TRYPSIN- AND CHYMOTRYPSIN-LIKE CDNAS FROM THE MIDGUT OF OSTRINIA NUBILALIS Abstract 135 Introduction 136 Experimental Procedures 138 Results and Discussion 142 References 151 CHAPTER 8. A (3-1,3-GALACTOSYLTRANSFERASE AND BRAINIAC/ BRE-5 HOMOLOG FROM OSTRINIA NUBILALIS Abstract 166 Introduction 166 Materials and Methods 169 Results and Discussion 175 References 180 CHAPTER 9. CONCLUSION 189 ACKNOWLEDGMENTS 198 V LIST OF TABLES Table 2.1. Mitochondrial codon usage table. 59 Table 2.2. Nucleotide frequencies in mitochondrial genomes. 59 Table 3.1. Twenty-six variable nucleotide positions observed between 79 14 North American O. nubilalis mitochondrial coxl and cox2 sequences. Table 3.2. Distribution of mitochondrial RFLP frequencies and 80 haplotypes in samples from 15 North American collection sites. Table 3.3. Mitochondrial AMOVA and modified ^-statistics. 81 Table 3.4. Significantly different pairwise Nei genetic distance (D) comparison 82 among mitochondrial haplotypes. Table 4.1. Ten Ostrinia nubilalis CA/GT and GA/CT microsatellite markers. 88 Table 5.1. North American O. nubilalis male ONZ1 genotypic, and heterogametic 103 female ONW1 and ONZ1 haplotype frequencies. Table 5.2. Male AMOVA and FsT, and female AMOVA and Ost values. 104 Table 6.1. Mendelian inheritance of cadherin alleles in O. nubilalis pedigrees 133 Ped31 and Ped62. Table 6.2. Mead, Nebraska O. nubilalis frequency of cadherin PCR-RFLP alleles. 134 Table 7.1. Ostrinia nubilalis trypsin- and chymotrypsin-like serine protease 162 gene primers. Table 7.2. Ostrinia nubilalis midgut cDNA clones and putative derived 163 mature serine protease properties. Table 7.3. Pair-wise similarity matrix of O. nubilalis midgut expressed 164 serine protease cDNA sequences. Table 7.4. Test of Mendelian inheritance of trypsin and chymotrypsin alleles 165 in O. nubilalis pedigrees PedlOb and Ped24a. VI LIST OF FIGURES Figure 2.1. Ostrinia mitochondrial genome map of sequenced regions. 58 Figure 2.2. Predicted tRNAARG secondary structures. 58 Figure 3.1. Location of 15 North American O. nubilalis collection sites. 77 Figure 3.2. Parsimony haplotype tree constructed from a 2,156 bp 78 mitochondrial DNA alignment. Figure 5.1. Sex linked microsatellite sequences. 102 Figure 6.1. Ostrinia nubilalis 1717 residue long cadherin-like peptide sequence. 125 Figure 6.2. Lepidopteran cadherin phytogeny. 127 Figure 6.3. Variation in lepidopteran cadherin toxin binding regions (TBRl and 2). 128 Figure 6.4. Alignment of O. nubilalis cadherin CrylA toxin binding region 1 129 (OnTBRl). Figure 6.5. Alignment of O. nubilalis cadherin CrylA toxin binding region 2 131 (OnTBR2). Figure 7.1. Trypsinogen and chymotrypsinogen-like protease sequences. 156 Figure 7.2. Lepidopteran trypsinogen and chymotrypsinogen peptide phytogeny. 159 Figure 7.3. Trypsin-(OnT25, OnT23, and OnT3) and chymotrypsin-like 161 (OnCl and OnC2) transcript reverse transcriptase (RT)-PCR detection among tissues. Figure 8.1. Peptide sequence alignment of invertebrate 185 (5-1,3-galactosyltransferases. Figure 8.2. A consensus (3-l,3-galactosyltransferase-5/brainiac/BRE-5 phytogeny. 186 Figure 8.3. Relative abundance O. nubilalis (3-1,3-galactosyltransferase 187 (Onb3GalT5) transcripts among larval tissues. Figure 8.4. Ostrinia nubilalis P-1,3-galactosyltransferase (Onb3GalT5) 188 5'-untranslated and promoter region sequence. vii ABSTRACT A candidate gene approach was taken to characterize Bacillus thuringiensis (Bt) crystalline {Cry) toxin resistance phenotypes of the European com borer, Ostrinia nubilalis (Hiibner), and anonymous genomic markers were used to estimate subpopulation differentiation and migration rates that may affect spread of resistance phenotypes in the North American population. Native and transgenic variants of Bacillus thuringiensis (Bt) crystalline {Cry) toxins control larval lepidopteran feeding upon important agronomic crop plants. Invertebrate Bt resistance phenotypes arise via altered expression of midgut proteases involved in Cry toxin activation (trypsins) and degradation (chymotrypsins), reduced toxin binding to N-acetylgalactoseamine (GalNAc) modified peptide receptors (cadherin, aminopeptidase, or alkaline phosphatase), or receptor glycosylation pathway knockouts (P- 1,3-galactosyltransferases; p3GalT5). Ostrinia nubilalis trypsin, chymotrypsin, cadherin, and p3GalT5 cDNA clones, transcript expression, and partial genomic DNA copies were characterized. Mendelian inheritance of trypsin, chymotrypsin, and cadherin genomic DNA markers suggest association between allele segregation and Bt resistance phenotypes can be conducted. Movement of O. nubilalis moths was indirectly estimated by genetic similarity between North American subpopulations using mitochondrial and genomic microsatellite markers. Marker data suggested differentiation between two O. nubilalis ecotypes, univoltine and bivoltine, which may affect movement of Bt resistance phenotypes in northern regions of the Midwest United States. 1 CHAPTER 1. GENERAL INTRODUCTION 1.1 Introduction Insect pests of cultivated crops cause economically detrimental damage to plant and yield worldwide. The European com borer, Ostrinia nubilalis, larvae are a major insect pest of com in Europe and North America. Introduction of transgenic maize expressing versions of the Bacillus thuringiensis (Bt) CrylAb toxin gene as a crop production option has raised concern for resistance development in target insects, similar to what historically has occurred with chemical pesticides. Evaluation of