Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1995 Analysis and application of the En transposable element and genetic study of resistance to Bipolaris maydis in maize Ru-Ying Chang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, and the Plant Pathology Commons Recommended Citation Chang, Ru-Ying, "Analysis and application of the En transposable element and genetic study of resistance to Bipolaris maydis in maize " (1995). Retrospective Theses and Dissertations. 10767. https://lib.dr.iastate.edu/rtd/10767 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]. INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI films the text directfy from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from aiQr type of conq)uter printer. The qnali^ of this reprodnction is dependent npon the qnali^ of the copy snbmitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard marging, and inqiroper alignment can adversefy affect reproduction. In the tmlikely event that the author did not send UMI 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. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to light in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are avaflable for ai^ photographs or illustrations appearing in this copy for an additional diaige. Contact UMI directty to order. A Bell & Howell Information Company 300 North 2eeb Road. Ann Artaor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Analysis and application of the En transposable element, and genetic study of resistance to Bipolaris maydis, in maize by Ru-Ying Chang A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Agronomy Interdepartmental Major: Genetics Approved: Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. Faffne Ihter^^artmental Major Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1995 DHI Number: 9540881 DMl Microform 9540881 Copyright 1995, by DHI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, HI 48103 ii TABLE OF CONTENTS GENERAL INTRODUCTION 1 Dissertation Organization 1 Objectives 2 Literature Review 4 1. DIFFERENTIAL INSERTIONS OF THE En TRANSPOSABLE ELEMENT ARE RELATED TO SOMATIC VARIEGATION PATTERNS AT THE MAIZE A2 LOCUS 15 Abstract 15 Introduction 16 Materials and Methods 17 Resiilts 19 Discussion 21 Acknowledgments 25 2. CHROMOSOME LABELING WITH TRANSPOSABLE ELEMENTS IN MAIZE 28 Abstract 28 Introduction 29 Materials and Methods 30 Results 31 Discussion 33 3. INSTABILITY OF THE MAIZE DISEASE-RESISTANCE GENE, Rpl,m TRANSPOSABLE ELEMENT-LADEN LINES 44 Abstract 44 Introduction 45 Materials and Methods 46 Results 48 Discussion 50 Acknowledgments 52 iii 4. ADDITIONAL RESULTS AND ANALYSIS IN Rpl TAGGING WITH TRANSPOSABLE ELEMENTS 56 Methods 56 Screening Resxilts 57 RFLP Analysis of Mutants 5 7 5. GENETIC CONTROL OF RESISTANCE TO BIPOLARIS MAYDIS: ONE GENE OR TWO GENES? 65 Abstract 65 Introduction 66 Materials and Methods 67 Results and Discussion 69 6. GENETIC EVIDENCE FOR DIGENIC RESISTANCE TO BIPOLARIS MAYDIS, RACE O, IN MAIZE 76 Recombination Test of the Two-Gene Model 76 An Alternative Explanation 79 Strategy for Molecular Marker Exchange Analysis 80 GENERAL SUMMARY 87 LITERATURE CITED 92 ACKNOWLEDGMENTS 104 1 GENERAL INTRODUCTION Dissertation Organization This dissertation is presented in an alternative format. It is composed of six chapters preceded by a general introduction and followed by a general summary, literature cited and acknowledgments. Chapter 1 is to be submitted for formal publication. Chapters 2,3 and 5 have been published in formal acadendc journals. Results presented in Chapters 2 and 3 were partly accomplished in my master's study. Chapters 4 and 6 are a continuation of Chapters 3 and 5, respectively. The first four parts are the analysis and application of the En transposable element and the last two parts are studies of resistance to Bipolaris maydis. Chapter 1 summarizes the restalts of a molecular analysis of "states" of the En transposable element. Three mutable alleles at the A2 locus in the anthocyanin pathway were studied for En insertion positions. These alleles contain an autonomous En, which shows a common spotting pattern when crossed to a common En tester allele al-m(r), and therefore have similar transposase expressions. A correlation is observed between En insertion positions in the single coding region of the intronless A2 gene and variegation patterns in the aleurone. Chapter 2 presents restalts of the chromosome labeling project. Because En has been shown to transpose more frequently to closely linked sites. En was relocated to each of the 20 maize chromosome arms by using reciprocal translocations. Arms containing an En element are said to be labeled by the element. The purpose of this project is to facilitate transposon tagging by close linkage of En to target genes. All 20 chromosome arms have been labeled. 2 Chapter 3 and Chapter 4 report results of transposon tagging ofRpl, a gene controlling resistance to common rust. In Chapter 3, a preliminary report of Rpl tagging is reported and instability of Rpl in different transposon-laden lines is compared. Additional results and more detailed RFLP analysis of some mutants are presented in Chapter 4. Chapter 5 reports the exceptional result in the rhm tagging project, rhm is a gene conferring resistance to southern com leaf blight (Bipolaris maydis). In contrast to the one-gene hypothesis, a two-gene model for resistance is proposed, and the rationale underlying this model is elucidated. Chapter 6 reports genetic tests of the two-gene model. These tests are consistent with the two-gene model. However, one last alternative has not been excluded. A molecular marker exchange test is proposed and will be conducted in the near future. Objectives Common rust and southern leaf blight are two important fungal diseases of maize in the United States as well as in other parts of the world (Shurtleff 1980). Common rust, caused by Puccinia sorghi, is common in temperate climates and has the potential to cause yield losses of susceptible maize (Hooker 1978), Southern leaf blight is wide-spread in tropical and subtropical areas. This disease caused the severe epiphytotic in 1970 in the United States with an estimated monetary loss of one billion dollars (Ullstrup 1978). Southern leaf blight is caused by a fungus originally named Helminthosporium maydis Nisikado and Miyake (Nisikado and Miyake 1926, cited in Ullstrup 1970), and later revised as Bipolaris maydis (Nisikado) Shoemaker (Alcorn 1983). 3 Several dominant resistance genes (Rp for resistance to P. sorghi) with numerous different alleles have been identified (Hooker 1978) for common rust. Resistance to southern leaf blight was determined to be controlled by a single recessive gene designated rhm for resistance to H. maydis (Smith and Hooker 1973). Transposable elements are segments of DNA capable of moving from one position to another in the genome (transposition). Since the elucidation of the transposition phenomena by McClintock (1946,1947), transposable elements have been an important area of research. Transposon tagging has been widely xised to clone genes after the initial exploitation of this approach in cloning the white locus in Drosophila (Bingham et al. 1981). Many genes have been cloned and molectalarly characterized in maize (Doring 1989; Gierl and Saedler 1992) as well as in other organisms. The objectives of this dissertation are: 1. to label all 20 chromosome arms of maize with the En transposable element; 2. to elucidate the basis of the 'state' of mutable pattern expression of three jB/i-mutable alleles at the intronless A2 locus; 3. to study instabiUty at, and transposon tag, the Rpl locus; 4. to analyse the inheritance of resistance to Bipolaris maydis. Results of the molecular analysis of En. states at the A2 locus and chromosome labeling are reported. Progress in transposon tagging of Rpl and the results of instability study of Rpl are presented. Evidence is also reported that supports a two-gene model for resistance to B. maydis instead of monogenic resistance documented earlier (Smith and Hooker 1973). 4 Literature Review Literature pertaining to chromosome labeling and resistance to Bipolaris maydis has been reviewed in depth in Chapters 2, 5 and 6. This aspect is not repeated in this review. Common rust Common rust is caused by the fungus Puccinia sorghi Schew. and is a disease affecting commercial field com in most parts of the world. Because of this, there has been intensive effort to examine the inheritance of resistance for several decades. This has contributed to the understanding of resistance to this disease and to the effort to secure this resistance gene for molecular analysis. A study of resistance to this disease was initiated in 1918 at Lafayette, Indiana (Mains 1931).