GENETIC ARCHITECTURE OF RESISTANCE TO PHYLOGENETICALLY DIVERSE IN

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Jose Luis Zambrano Mendoza, M.S.

Graduate Program in Horticulture and Crop Science

The Ohio State University

2013

Dissertation Committee:

Professor David M. Francis, Advisor

Professor Margaret G. Redinbaugh, Advisor

Professor Leah K. McHale

Professor Peter R. Thomison

Copyrighted by

Jose Luis Zambrano Mendoza

2013

Abstract

Virus diseases of maize can cause severe yield reductions threatening crop production and food supplies in some regions of the world. Genetic resistance to some of the major diseases has been characterized. Previously, resistance loci for Maize dwarf , (MDMV), mosaic virus (SCMV), Wheat streak mosaic virus (WSMV), all members of the ; and Maize chlorotic dwarf virus

(MCDV) and Maize mosaic virus (MMV), were located to maize map bins 3.04 / 3.05,

6.01, and 10.05 using diverse mapping populations and different environments. For other diseases, including those caused by Maize rayado fino virus (MRFV), Maize fine streak virus (MFSV) and Maize necrotic streak virus (MNeSV), nothing was known about genetic resistance. The main goals of the research reported in this dissertation were to identify the genetic location and mode of inheritance of or quantitative trait loci

(QTL) conferring resistance to MRFV, MNeSV, and MFSV and to determine whether they are found in the same regions of the maize containing resistance to the

Potyviridae and the other viruses.

MRFV causes one of the most important virus diseases of maize in the Americas.

Genetic resistance was previously identified in a tropical highland inbred line and two tropical landraces. The fact that the landraces were populations complicated genetic analysis of the resistance. In this research, novel sources of resistance to MRFV were

ii identified, including inbred lines Oh1VI, Cuba, CML287, Ki11 and CML333. The discovery of novel sources of resistance in maize inbred lines facilitated the identification of a QTL conferring resistance to MRFV and will make possible its incorporation and use into breeding programs.

The maize line, Oh1VI, is resistant to multiple viruses. To characterize multiple virus resistance in this line, a maize recombinant inbred line (RIL) population, and a number of F1 plants and F2 populations derived from a cross of Oh1VI and the virus- susceptible inbred line Oh28 were genotyped and screened for their responses to:

MDMV, SCMV, WSMV, MCDV, MFSV, MMV, MRFV and MNeSV. A genetic map containing 256 markers distributed among 10 linkage groups representing the maize chromosomes was built using genotypic information derived from 768 SNP multiplex assay from the Illumina® BedArray™ platform and 21 informative (polymorphic) SSRs.

Composite interval mapping identified 19 significant associations between regions of the genome and resistance to the eight viruses tested. Of these, 15 were clustered on chromosomes 6, 3, and 10. An additional novel cluster of virus resistance QTLs were found in chromosome 2. In Oh1VI, most of the resistance appeared to be dominant and segregation of resistance to specific virus in F2 plants was consistent with one to three resistance genes. It is unknown whether these regions of clustered QTLs contain single or multiple virus resistance genes, but the linkage of genes conferring resistance to multiple virus diseases in this population could facilitate breeding efforts to develop multi-virus resistant maize. Agronomic information of the RIL population and selected multi-virus resistance lines in absence of viral diseases is also presented.

iii

Dedicated to my family

iv

Acknowledgments

I am grateful to my advisers Dr. Margaret Redinbaugh and Dr. David Francis for their intellectual guidance, discussion and constant support for this project and my academic courses. Their assistance in editing this dissertation is also greatly appreciated.

I thank my advisory committee members, Dr. Leah McHale and Dr. Peter Thomison for their advice during the course of my graduate study. I also want to thank Mark Jones,

Jane Todd, Kristen Willie, Christopher Nacci, Katia Morales, H. Miao, Rafael Diniz and

Erick Brenner, who are/were members or interns of the USDA, ARS Corn, Soybean and

Wheat Quality Research Unit (CSWQRU) in Wooster, for technical assistance and friendship. I wish to also thank Dr. Ray Louie and Dr. Lucy Stewart for valuable discussion and encouragement. I thank my laboratory partners Fiorella Cisneros and

Bryan Cassone for their help and friendship. I thank my wife Katia for her moral support, patience, company and encouragement during my studies and research. I want to thank

Adriana Thomas of DuPont Agricultural Biotechnology and the DuPont Pioneer Marker

Laboratory, Johnston, IA for conducting the genotyping of the maize population that was a key addition to this research. I want to thank the Republic of Ecuador and the Instituto

Nacional de Investigaciones Agropecuarias for providing a fellowship for my PhD study.

This research was supported by the USDA, ARS, CSWQRU.

v

Vita

1999...... Ingeniero Agropecuario, Escuela Politécnica

del Ejercito, Sangolquí, Ecuador

1999-2005 ...... Research Assistant, Instituto Nacional de

Investigaciones Agropecuarias (INIAP),

Programa de Maíz, Estación Experimental

Santa Catalina, Quito, Ecuador

2005...... Visiting Scholar, Department of Plant

Pathology, The Ohio State University

2006-2007 ...... M.S. Plant Sciences, Wageningen

University

2008-2010 ...... Research Leader, INIAP, Programa de Maíz,

Estación Experimental Pichilingue,

Quevedo, Ecuador

2010 to present ...... Graduate Student, Department of

Horticulture and Crop Science, The Ohio

State University

vi

Publications

1. Zambrano, J.L., M.D. Francis, and M.G. Redinbaugh. 2013. Identification of resistance to Maize rayado fino virus in maize inbred lines. Plant Dis., in press.

Fields of Study

Major Field: Horticulture and Crop Science

vii

Table of Contents

Abstract ...... ii Acknowledgments...... v Vita ...... vi Table of Contents ...... viii List of Tables ...... xiii List of Figures ...... xv

Chapter 1: Introducction and literature review ...... 1

1.1. Introduction ...... 1 1.2. Objectives ...... 3 1.3. Literature review ...... 4 1.3.1. Plant disease resistance ...... 4 1.3.2. Qualitative disease resistance ...... 5 1.3.2.1. Plant basal disease resistance ...... 5 1.3.2.2. R- mediated disease resistance ...... 6 1.3.3. Quantitative disease resistance ...... 9 1.3.3.1. QTL mapping ...... 10 1.3.4. Plant resistance to virus diseases ...... 12 1.3.5. Mechanisms of virus resistance ...... 16 1.3.6. Genetics of virus resistance in maize ...... 19 1.3.6.1. Potyviridae (MDMV, SCMV, and WSMV) ...... 20 1.3.6.2. (MMV and MFSV) ...... 21 1.3.6.3. (MRFV) ...... 22 1.3.6.4. (MCDV) ...... 24 viii

1.3.6.5. (MNeSV) ...... 25 1.3.7. Clustering of viral resistance genes in maize ...... 26 1.3.8. Breeding for virus resistance in maize ...... 28 1.4. Preview of thesis ...... 30 1.5. References ...... 32

Chapter 2: Identification of resistance to Maize rayado fino virus in maize inbred lines ...... 43

2.1. Abstract ...... 43 2.2. Introduction ...... 44 2.3. Materials and methods ...... 46 2.3.1. Maize germplasm ...... 46 2.3.2. Virus isolate and insect vector ...... 46 2.3.3. Evaluation of maize responses to MRFV ...... 47 2.3.4. Symptom evaluation ...... 47 2.3.5. MRFV detection with ELISA ...... 48 2.3.6. Experimental design and data analysis ...... 49 2.3.7. Inheritance of resistance ...... 49 2.4. Results ...... 50 2.4.1. Responses of maize accessions to inoculation with MRFV ...... 50 2.4.2. Inheritance of resistance to MRFV in Oh1VI ...... 52 2.5. Discussion ...... 53 2.6. Acknowledgments...... 57 2.7. References ...... 58

Chapter 3: Genetic analysis of resistance to six virus diseases in a multiple virus- resistant maize inbred line ...... 68 3.1. Abstract ...... 68 3.2. Introduction ...... 69 3.3. Materials and methods ...... 72 3.3.1. Plant material ...... 72 3.3.2. Viruses and vectors...... 73 ix

3.3.3. Inheritance of the resistance...... 74 3.3.4. RIL phenotypic analysis ...... 76 3.3.5. RIL Genotypic analysis ...... 77 3.3.6. Linkage and mapping analysis ...... 78 3.4. Results ...... 79 3.4.1. Inheritance of the resistance...... 79 3.4.2. RIL phenotypic analysis ...... 80 3.4.3. Linkage Map ...... 82 3.4.4. QTL mapping for virus resistance ...... 82 3.4.5. Interaction between QTLs ...... 84 3.5. Discussion ...... 85 3.6. Acknowledgments...... 91 3.7. References ...... 93

Chapter 4: QTL mapping of resistance to Maize rayado fino virus ...... 108

4.1. Abstract ...... 108 4.2. Introduction ...... 109 4.3. Materials and methods ...... 110 4.3.1. Plant material ...... 110 4.3.2. Vector and virus ...... 110 4.3.3. Disease evaluation ...... 111 4.3.4. Analysis of phenotypic data ...... 112 4.3.5. Molecular Data...... 112 4.3.6. QTL analysis ...... 113 4.4. Results ...... 113 4.5. Discussion ...... 115 4.6. Acknowledgments...... 118 4.7. References ...... 119

x

Chapter 5: Identification of a major quantitative trait loci controlling resistance to Maize necrotic streak virus in maize using a selective mapping strategy ...... 128

5.1. Abstract ...... 128 5.2. Introduction ...... 129 5.3. Materials and methods ...... 131 5.3.1. Maize germplasm ...... 131 5.3.2. Virus isolate and inoculum ...... 132 5.3.3. Virus inoculations ...... 132 5.3.4. Evaluation of disease symptoms ...... 134 5.3.5. Selective mapping and phenotyping of RIL populations ...... 134 5.3.6. Analysis of RIL phenotypic data ...... 136 5.3.7. QTL analysis ...... 136 5.4. Results ...... 137 5.4.1. Inheritance of the resistance...... 137 5.4.2. Selective mapping and phenotyping of RIL populations ...... 138 5.4.3. QTL mapping ...... 139 5.5. Discussion ...... 140 5.6. Acknowledgments...... 146 5.7. References ...... 147

Chapter 6: Agronomic evaluation of selections from a maize recombinant inbred line population with multi-virus resistance ...... 161

6.1. Abstract ...... 161 6.2. Introduction ...... 162 6.3. Material and methods ...... 163 6.3.1. Plant material ...... 163 6.3.2. Agronomic evaluation of the RIL population ...... 164 6.3.3. Selection of multi-virus resistant lines ...... 165 6.3.4. Agronomic evaluation of the multi-virus resistance lines ...... 166 6.4. Results ...... 167 6.4.1. Agronomic evaluation of the RIL population ...... 167 xi

6.4.2. Selection of multi-virus resistant lines ...... 167 6.4.3. Agronomic evaluation of the multi-virus resistance lines ...... 168 6.5. Discussion ...... 169 6.6. Acknowledgments...... 172 6.7. References ...... 173

Chapter 7: Conclusions ...... 180

7.1. Conclusions ...... 180

Bibliography ...... 187

xii

List of Tables

Table 2.1. Responses of maize accessions to Maize rayado fino virus (MRFV) inoculation………………………………………………………………………………..62

Table 2.2. Inheritance of resistance to Maize rayado fino virus (MRFV) in F1 and F2 maize populations derived from the resistant Oh1V1 and the susceptible Oh28………..64

Table 3.1. Inheritance of maize resistance to six viruses in F1 and F2 generations derived from the resistant inbred line Oh1VI and the susceptible Oh28……………………..…101

Table 3.2. Components of variance for disease incidence and area under the disease progress curve (AUDPC) for a maize recombinant inbred line (RIL) population inoculated with five viruses ………………….…………………………………….…..102

Table 3.3. Location and genetic effects of QTLs associated with virus resistance in maize inbred line Oh1VI………………………………………………………………………103

Table 4.1. Components of variance for disease incidence and area under the disease progress curve in two maize recombinant inbred line populations inoculated with Maize rayado fino virus…………………………………………………………….………….123

Table 4.2. Location and genetic effects of QTLs associated with resistance to Maize rayado fino virus in two recombinant inbred line (RIL) populations of maize…………………………………………………..………………..…..…………..124

Table 5.1. Segregation of resistance to Maize necrotic streak virus in F1 and F2 progenies………………………………………………………………………………..152

Table 5.2. Components of variance for area under the disease progress curve and disease incidence in two maize recombinant inbred line populations inoculated with Maize necrotic streak virus…………………………………………………………………….153

Table 5.3. Location and genetic effects of a major quantitative trait locus associated with resistance to Maize necrotic streak virus identified by composite interval mapping in a maize recombinant inbred line population derived from a Oh1V1 x Oh28 cross……...154 xiii

Table 5.4. Location and genetic effects of markers associated with resistance to Maize necrotic streak virus identified by single marker regression in a maize recombinant inbred line population derived from a Oh1VI x Va35 cross……………………………155

Table 6.1. Descriptive statistics for five agronomic traits measured in 256 maize recombinant inbred lines derived from Oh1VI and Oh28 evaluated during the summers of 2011 and 2012 at Snyder Farm (OARDC, Wooster, OH)…...…………...………….…176

Table 6.2. Pearson correlation coefficients and probabilities for multi-virus resistance index and five agronomic traits evaluated in 256 maize recombinant inbred lines during the summers of 2011 and 2012 at Snyder Farm (OARDC, Wooster, OH)…………….177

Table 6.3. Means for five agronomic traits of selected multi-virus resistant maize inbred lines derived from Oh1VI and Oh28 evaluated during the summers of 2011 and 2012 at Snyder Farm (OARDC, Wooster, OH)…………………………………………………178

Table 6.4. Yield and other agronomic traits of 13 maize inbred lines evaluated during summer 2012 at Snyder Farm (OARDC, Wooster, OH)……………………………….179

Table 7.1. Viruses of maize discussed in this dissertation and mode of inheritance of the resistance in the maize inbred line Oh1VI……………………………………………...185

xiv

List of Figures

Fig. 2.1. Symptom severity scale used to evaluate the response of maize seedlings to inoculation with Maize rayado fino virus………………………………………………..65

Fig. 2.2. Maize rayado fino virus disease progress in susceptible maize accessions. Symptoms were evaluated in maize varities Spirit and B73 at 7, 14, and 21 days post inoculation (dpi). Data presented are the means ± s.d. for three replications with 10 plants per replication. A, Incidence and. B, Mean severity rating of symptomatic plants……...66

Fig. 2.3. Maize rayado fino virus antigen in maize leaves estimated with semi- quantitative PAS-Enzyme-linked immunosorbent assay. Titer was estimated in selected maize lines at 21 days post inoculation. Data presented are the mean ± s.d. Bars labeled with same letter are not significantly different (Tukey test, P = 0.05)…………………..67

Fig. 3.1. Symptom severity scale for: A, Maize mosaic virus; B, Maize fine streak virus; and C, Maize chlorotic dwarf virus. Where, 1 = no disease symptoms, 3 = mild or incomplete symptoms and 5 = severe symptoms……………………………………….104

Fig. 3.2. Mean distribution of symptom incidence among 256 maize RILs inoculated with Maize mosaic virus (MMV), Maize fine streak virus (MFSV), Maize chlorotic dwarf virus (MCDV), Wheat streak mosaic virus (WSMV), Maize dwarf mosaic virus (MDMV) and (SCMV) evaluated between 14 and 21 days post inoculation………………………………………………………………………………105

Fig. 3.3. Mean distribution of area under the disease progress curve (AUDPC) scores for 256 maize RILs inoculated with Maize mosaic virus (MMV), Maize fine streak virus (MFSV) and Maize chlorotic dwarf virus (MCDV) evaluated at 21 days post inoculation…………………………………………………………………..………….106

Fig. 3.4. Genetic location of maize QTLs conferring resistance to virus diseases. The bars indicate significant LOD scores (p < 0.01) across the Oh1VI x Oh28 genetic map (cM) for: a, Maize chlorotic dwarf virus (MCDV); b, Maize mosaic virus (MMV); c, Maize fine streak virus (MFSV); d, Maize dwarf mosaic virus (MDMV); e, Sugarcane mosaic virus (SCMV); and f, Wheat streak mosaic virus (WSMV). The fine gray circle within the band for each virus indicates the significance threshold for LOD scores. The ribbons xv link the regions where QTL interactions for some virus diseases were detected (p < 0.0001)………………………………………………………………………………….107

Fig. 4.1. Maize rayado fino virus disease progress in susceptible controls and parents of the maize RIL populations Ki11 x B73 and Oh1VI x Oh28 at 7, 14, and 21 days post inoculation. Data presented are the means ± s.d. for three replications with 10 plants per replication. A, Incidence and B, Mean severity rating of symptomatic plants using a rating scale of 0 to 5, where 0 = no disease symptoms and 5 = dead…………………..125

Fig. 4.2. Means for AUDPC and incidence of Maize rayado fino virus at 21 days post inoculation in A) 256 maize RIL population derived from Oh1VI x Oh28, and B) 193 maize RIL population derived from Ki11 x B73……………………………………….126

Fig. 4.3. Location (cM) of a quantitative trait loci (QTL) for resistance to Maize rayado fino virus on chromosome 10 of maize estimated from the Oh1VI x Oh28 RIL population (left) and Ki11 x B73 RIL population (right). Horizontal dot lines denotes markers present in both genetic maps. Dashed and black blocks indicates the genome region covered by the QTL at P < 0.001 based on permutations in the Oh1VI xOh28 and Ki11 x B73 maps, respectively…………………………………………………………………127

Fig. 5.1. Leaf of maize seedlings inoculated with Maize necrotic streak virus. In the 1 to 5 scale, 1 = no symptoms, 2 = mild symptoms, 3 = intermediate, 4 = moderately severe symptoms, and 5 = severe disease symptoms…………………………………………..156

Fig. 5.2. Maize necrotic streak virus disease incidence in maize lines Oh1VI, Oh28, and their F1 and F2 generations. Disease incidence was evaluated at 9, 16, and 23 days post inoculation as the percentage of plants showing symptoms. Data presented is the mean + standard error of 5, 5, 6, and 13 replications for Oh1VI, Oh28, F1 and F2, respectively……………………………………………………………………………..157

Fig. 5.3. Maize necrotic streak virus disease progress in maize lines Oh1VI, Oh28, and their F1 and F2 generations. Severity of symptoms were evaluated at 9, 16, and 23 days post inoculation using a rating scale from 1 to 5, where 1 = no disease symptoms and 5 = severe symptoms. Data presented is the mean ± standard error of plants that showed symptoms……………………………………………………………………………….158

Fig. 5.4. Mean distribution of disease severity and area under the disease progress curve (AUDPC) at 23 days post inoculation evaluated on: A) 92 maize recombinant inbred lines (RIL) derived from Oh1VI x Oh28, and B) 105 maize RIL derived from Oh1VI x Va35 for reaction to Maize necrotic streak virus………………….……………………159

Fig. 5.5. Location (cM) of a quantitative trait loci conferring resistance to Maize necrotic streak virus (MNeSV) mapped on chromosome 10 of the maize recombinant inbred line population Oh1VI x Oh28. Loci conferring resistance to Wheat streak mosaic virus xvi

(WSMV), Maize dwarf mosaic virus (MDMV), and Maize rayado fino virus (MRFV) previously mapped in the same population are also shown. The relative map position of mcd2, a locus conferring resistance to Maize chlorotic dwarf virus (MCDV), is shown to the right…………………………………………………………………………………160

Fig. 7.1. Genetic distribution of QTLs conferring resistance to Maize chlorotic dwarf virus (MCDV), Maize mosaic virus (MMV), Maize fine streak virus (MFSV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV), Maize rayado fino virus (MRFV), and Maize necrotic streak virus (MNeSV) in the maize recombinant inbred line population Oh1VI x Oh28. Bars indicate LOD scores from composite interval mapping across the genetic map (cM) for each virus disease. Lines within the ring for each virus indicate a significance threshold for LOD scores of P < 0.01………………………………………………………………………186

xvii

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1. Introduction

Plant viruses are important agricultural that cause significant economic losses through yield and quality reduction (Ali and Yan, 2012; Gomez et al., 2009; Kang et al., 2005). Maize dwarf mosaic virus (MDMV) was the first reported to cause significant yield reduction in the United States corn crop (Williams and Alexander,

1965; Gordon et al., 1981). First observed in Ohio (1962), epiphytotics of MDMV occurred in Texas (1968), Maryland (1974), Ohio and New York (1976), and Minnesota

(1977). By 1978 the disease had been reported in 41 states, causing yield reductions up to

72% (Gordon et al., 1981; Boothroyd 1981). Contemporary commercial hybrids of in the United States have genetic resistance to MDMV, but the virus is still a problem in production (Williams and Pataky, 2012). MDMV and other members of the Potyviridae remain among the most damaging diseases of maize in some regions of the world, including South America, China, and Africa, where the virus is present as a disease complex known as maize lethal necrosis (Castillo and Hebert, 1974;

Uyemoto et al., 1980; Wangai et al., 2012).

1

Although viruses are relatively simple genetic entities, the mechanisms by which disease symptoms are generated and how plants resist these affects are largely unknown

(Kang et al., 2005). The genetics of maize resistance to MDMV and related viruses in the

Potyviridae has been described (Jones et al., 2007; Jones et al., 2011; McMullen and

Louie, 1991; McMullen et al., 1994; Redinbaugh and Pratt, 2009; Redinbaugh et al.,

2004; Stewart et al., 2012).For many other viruses for which infection or transmission is complex, there has been a lack of definitive information regarding sources of resistance, the mode of inheritance of the resistance and the loci or genes which confer resistance.

Some of these viruses include Maize rayado fino virus (MRFV), Maize necrotic streak virus (MNeSV) and Maize fine streak virus (MFSV).

MRFV incites one of the most important virus diseases (rayado fino or achaparramiento) of maize in Central and South America (De-Oliveira et al., 2004;

Gamez and Saavedra, 1986; Gimenez-Pecci et al., 2000; Gordon and Thottappilly, 2003;

Kogel et al., 1996; Valdez et al., 2004; Vasquez and Mora, 2007). Genetic resistance to

MRFV seems to be rare as fewer than 3% of accessions tested showed reduced or no disease symptoms (Bustamante et al., 1998; Ramirez-Rojas et al., 1998; Toler et al.,

1985; Vandeplas, 2003). The identification of inbred lines with resistance to MRFV and loci conferring resistance to MRFV will allow breeders to introgress resistance into elite breeding material, alleviating the losses caused by this disease.

QTL mapping studies of viruses in the Potyviridae have provided information about the genetic architecture of maize resistance. Major genes conferring resistance to the Potyviridae have been found in clusters on regions of maize chromosomes 1, 3, 6 and

2

10 (Di Renzo et al., 2004; Jones et al., 2004; Jones et al., 2007; Kyetere et al., 1999;

McMullen and Louie, 1991; McMullen et al., 1994; Redinbaugh and Pratt, 2009;

Redinbaugh et al., 2004; Stewart et al., 2012; Welz et al., 1998); however, a number of virus resistance QTLs have been identified on other regions of the maize genome

(Bonamico et al., 2012; Di Renzo et al., 2004; Dintinger et al., 2005; Kyetere et al., 1999;

Martin et al., 2009; Pernet et al., 1999a; Pernet et al., 1999b; Rufener et al., 1996; Wang et al., 2007a; Welz et al., 1998). With the exception of resistance to viruses in the

Potyviridae and a few viruses from other families that have been relatively well studied, little is known about the number and location of genes conferring resistance to other virus diseases, including but not limited to MRFV, MNeSV, and MFSV (Redinbaugh and

Pratt, 2009). Therefore, further investigation is needed to map the location of the genes conferring resistance to these diseases, and to clarify weather resistance to different virus clusters in the maize genome. A better understanding of resistance to virus pathogens in maize could facilitate the genetic control of these important diseases in many parts of the world, and will contribute to the knowledge of the plant-virus interaction.

1.2. Objectives

Previously, resistance loci for members of the Potyviridae including MDMV,

Sugarcane mosaic virus (SCMV) and Wheat streak mosaic virus (WSMV), and the

Secoviridae Maize chlorotic dwarf virus (MCDV) and the Rhabdoviridae Maize mosaic virus (MMV) were mapped to maize genetic bins 3.04 / 3.05, 6.01, and 10.05 using diverse mapping populations and different environments (Ding et al., 2012; Dussle et al.,

3

2000; Ingvardsen et al., 2010; Jones et al., 2004; Jones et al., 2007; Jones et al., 2011;

McMullen et al., 1994; Ming et al., 1997; Prazeres De Souza et al., 2008; Wu et al., 2007;

Zhang et al., 2003). The main goals of the research reported in this dissertation were to identify the genetic location of genes or QTLs conferring resistance to MRFV, MNeSV, and MFSV and to determine whether they are found in the same maize regions of the genome containing resistance to the Potyviridae and other viruses. The specific objectives of this research project were:

1) Identify sources of resistance to MRFV in a diverse set of public maize inbred lines that allow further genetic analysis and selection.

2) Map the genetic location of genes or QTLs that confer resistance to MRFV,

MFSV, MNeSV, MDMV, SCMV, WSMV, MCDV and MMV, and characterize multiple virus resistance in a maize RIL population.

3) Select genotypes within the RIL population with multiple virus disease resistance and superior agronomic traits that could be used for applied breeding purposes.

1.3. Literature review

1.3.1. Plant disease resistance

Plant diseases can drastically reduce crop yield, threatening food security (Ali and

Yan, 2012). Crop losses caused by diseases worldwide are estimated to be worth around

$220 billion annually (Agrios, 2005). Plant resistance is the most cost-effective and environmentally friendly approach to control diseases (Gururani et al., 2012). A large number of plant resistance genes have been characterized and efficiently used in crop

4 breeding programs (Ali and Yan, 2012; Gururani et al., 2012). However, global yield losses due to diseases remains significant. Therefore, a challenge in plant biology remains to identify new resistance, clarify the genetic mechanisms, and efficiently incorporate resistance genes to alleviate existing and emerging problems.

To control diseases, plants have evolved pre- and post- invasion resistance mechanisms controlled by genes whose products directly or indirectly detect specific pathogen effectors and trigger plant defense responses (Chisholm et al., 2006).

Two general types of disease resistance have been recognized in plants based on genetics: qualitative, complete or major gene resistance usually conditioned by single genes (R gene), and quantitative, incomplete, or multi-gene resistance conditioned by many genes of small effect, known as quantitative trait loci (QTL) (Poland et al., 2009). Despite the clear theoretical difference between quantitative and qualitative disease resistance, it is clear that pure qualitative and pure quantitative plant disease resistance are two ends of the same continuum, and that most resistance genes reside between these two extremes

(Balint-Kurti and Johal, 2009).

1.3.2. Qualitative disease resistance

1.3.2.1. Plant basal disease resistance

Basal resistance constitutes the first line of plant defense to a wide range of pathogens and is closely associated with non-host resistance (Gururani et al., 2012). Non- host resistance is the most common form of plant resistance and is highly effective against a range of potentially pathogenic microorganisms. It is defined as resistance of an

5 entire plant species to all isolates and races of a specific pathogen species (Ali and Yan,

2012). Although the molecular mechanisms behind non-host resistance are only emerging, it has been accepted that both constitutive cellular barriers and inducible responses constitute the basis of this form of plant resistance (Mysore and Ryu, 2004).

The plant cuticle, wall, , actin microfilaments, and phytoanticipins have been described as a passive mechanism providing the first defense against pathogen invasion (Guest and Brown, 1997). The second obstacle an invading pathogen faces are the inducible plant defenses. Phytoalexins, plant hormones (i.e. ethylene and salicylic acid), wound-induced protein kinase (WIPK), salicylic acid-induced protein kinase

(SIPK), and heat shock proteins (Hsps) are induced after pathogen attack, and have been found to play a crucial role in non-host resistance (Kanzaki et al., 2003; Mysore and Ryu,

2004; Zhang and Klessig, 2001). Inducers of basal defense are often conserved microbial elicitors produced by pathogens, such as bacterial flagellum or fungal chitin. These molecules are known as pathogen-associated molecular patterns (PAMPs) and are recognized by host receptor proteins called pattern recognition receptors (PRRs).

Stimulation of PRRs leads to plant triggered immunity (PTI) (Ali and Yan, 2012; Boller and Felix, 2009; Mysore and Ryu, 2004).

1.3.2.2. R-gene mediated disease resistance

Strong similarities exist between some non-host and host resistance responses and it is not clear if they share some of the same molecular mechanisms (Mysore and Ryu,

2004). R gene mediated resistance is perhaps the best understood mechanism of

6 resistance. It provides a rapid and effective response to pathogen attack and limits further infection and spread of the disease (Gururani et al., 2012). This response involves host receptor proteins that recognize pathogen virulence molecules called effectors, encoded by Avr (avirulence genes) that are delivered into host cells at the beginning of infection.

This resistance mechanism is known as the ‘gene-for-gene’ model, which states that for a plant to be resistant to a pathogen there must be matching pairs of resistance genes (R) and Avr in the host and pathogen, respectively (Flor, 1971). This gene for gene interaction is interpreted as the receptor-ligand model which states that the R gene protein directly recognizes the ligands specified by the bacterial Avr genes (Bonas and Lahaye

2002). The recognition of the R and Avr products induces an effector-triggered immunity

(ETI) response in the plant, leading to the activation of a signal transduction cascade responsible for the activation of local and systemic defense response. ETI results in disease resistance and usually in a hypersensitive response (HR), a form of programmed cell death at the infection site, and/or the production of antimicrobial metabolites (Jones and Dangl, 2006). Because there is little evidence for direct interaction between the R and

Avr proteins, an alternative hypothesis known as the ‘guard hypothesis’ was proposed

(Dangl and Jones, 2001; van der Biezen and Jones, 1998). It states that resistance (HR and/or production of antimicrobial metabolites) is initiated when an R protein detects an attack of its guardee, and does not necessarily involve direct interactions between the R and Avr proteins (Gururani et al., 2012; McDowell and Woffenden, 2003). The ‘guard hypothesis’ implies that R proteins indirectly recognize pathogen effectors (Avr proteins) by monitoring the integrity of the effector target (Mansfield, 2009).

7

A large number of plant pathogen effectors have been described. They often mimic plant hormones or inhibit plant cellular functions what contribute to pathogen virulence. Effectors can operate either in the extracellular matrix or inside host cells. For instance, the tomato R genes Cf2, CF4, Cf5 and Cf9 recognize specifically extracellular effectors produced by Cladosporium fulvum, and the Arabidopsis RPP13 R gene recognize the pathogen effector Atr13 from Hyaloperonospora parasitica inside the host cell. (Jones and Dangl 2006). If an effector is recognized by the host, resistance occurs, and the recognized effector is named as an avirulence (Avr) protein. Lack of recognition causes disease and the effector is named as a virulence (avr) protein. Most of the R genes recognize pathogen effectors indirectly, although there are few examples of direct interaction (Mansfield, 2009).

More than 50 R genes have been cloned and characterized from various plant species (Wang et al., 2007b). Interestingly, most of their protein motifs have some features in common, suggesting similar mechanisms underlying disease resistance and/or evolution. The conserved motifs include leucine-rich repeat (LRR), nucleotide-blinding site (NBS), a mammalian interleukin-1 receptor (TIR), a coiled coil (CC) structure, transmembrane domains (TM) and protein kinase domain (PK). Using these and other features, plant disease resistance genes have been classified into seven groups (Gururani et al., 2012). The first major class of R genes encodes cytoplasmic proteins that have a

NBS, a C-terminal LRR and a putative CC at the N-terminus. The second class of resistance genes encodes cytoplasmic proteins that have LRR, NBS motifs and an N- terminal domain with homology to the mammalian TIR domain. The third major class of

8 plant resistance genes encodes extra cytoplasmic leucine rich repeats (eLRR) attached to a transmembrane domain (TrD). The fourth class of R genes encodes proteins that consist of an extracellular LRR domain, a TrD domain, and an intracellular serine-threonine kinase (KIN) domain. The fifth class encodes proteins that contain a TrD domain fused to a putative CC domain. The sixth class of resistance genes encodes proteins that have TIR,

NBS, LRR, and a C-terminal extension with a putative nuclear localization signal (NLS) and a WRKY domain. The WRKY domain is a 60 amino acid region that contains conserved amino acid sequence WRKYGQK at its N-terminal end with a zinc-finger-like motif. The seventh major class of resistance genes consists of enzymatic R genes that do not encode LRR or NBS domains (Gururani et al., 2012). This type of gene (i.g. maize

Hm1) encodes an enzyme that inactivates pathogen toxins in the host (Johal and Briggs,

1992). From studies in plants and animals, these domains are responsible for protein- protein recognition, and their suggest related mechanism and evolution.

1.3.3. Quantitative disease resistance

Quantitative resistance is conferred by many genes with small effects. It is generally assumed to be non-race specific (though exceptions exist) and provides intermediate to high levels of resistance (Balint-Kurti and Johal, 2009). Plants with quantitative resistance show continuous levels of resistance controlled by multiple genes or quantitative trait loci (QTL) with a large environmental influence (Michelmore, 1995).

The molecular mechanisms of quantitative resistance have not been well characterized due to the incomplete and inconsistent nature of the resistant phenotype (Poland et al.,

9

2009). In addition, gene by gene (epistasis) and gene by environment interactions play an important role in the phenotypic expression of QTLs complicating fine mapping and cloning approaches (Ali and Yan, 2012). It is hypothesized that quantitative disease resistance is conditioned by many genes regulating morphological and developmental phenotypic stages, or that quantitative resistance is conferred by partially defeated R genes that slow down disease development (Poland et al., 2009).

Although various authors have speculated on the types of genes behind QTLs, there is not a clear molecular difference between a major R gene and a QTL. High resolution mapping studies and bioinformatics have often found R genes in QTL regions

(Kump et al., 2011; Poland et al., 2011; Wisser et al., 2006; Xiao et al 2007). These results suggest that the name (QTL or gene) is reflective of the Mendelian or biometric approach used for the identification and how the trait was measured.

Environment can affect the expression and efficiency of a gene or QTL. Genetic and environment interaction occurs when the basic phenotypic additive model (phenotype

= genotype + environment) fails. The additive model implies that the differences between genotypes remain the same across environments (van Eeuwijk 2006). In fact, resistance as any other phenotype is highly dependent of environmental stimuli, including temperature, nutrients, water, light, etc., and developmental time.

1.3.3.1. QTL mapping

The integration of molecular biology and quantitative genetics has provided valuable tools, methods and approaches to understand the genetic mechanisms of

10 quantitative traits. QTL mapping is the term used to describe the use of molecular markers to determine the genetic location of the genes or loci responsible for a trait with quantitative inheritance (Chahal and Gosal, 2002). The information provided by diverse plant transcriptome and genome sequencing projects has facilitated the development of molecular markers based on DNA polymorphisms that are well distributed through the entire genome (Hamilton and Buell, 2012). Sequencing technology combined with highly parallel genotyping platforms have increased access to high throughput molecular marker technology for translational research. Several papers published over the last two decades have provided valuable information on the genetic architecture of disease resistance such as the number, location, and action of genome regions conditioning the trait (Wisser et al., 2006; Young, 1996). For instance, Wisser et al. (2006) summarized the organization of disease resistance in maize. They looked at the genetic locations of 17 R genes and 437 resistance QTLs and found evidence for clustering of R genes and disease resistance

QTLs in several regions of the genome. Additionally, they found several chromosomes segments that were associated with resistance to multiple diseases caused by taxonomically diverse pathogens. Genetic map bins 6.01, 3.04, and 3.05 (based on the

IBM2 map) were noteworthy due to the number of resistances clustering there. Multiple disease resistance occurs when the same locus confers resistance to multiple pathogens

(Ali and Yan, 2012). The multiple disease resistance loci found on bins 3.04 and 3.05 was recently confirmed by Zwonitzer et al. (2010) using a RIL population derived from

Ki14 x B73. The clustering of multiple diseases resistance could be the effect of genes showing pleiotropic effects for multiple diseases (Poland et al., 2009; Wisser et al.,

11

2006). We also known R genes cluster due to expansion and divergence of multi-gene families. Studies conducted in potato, rice, lettuce, coffee, among others, have also found evidence for clustering of genes conferring quantitative and qualitative disease resistance

(Gebhardt and Valkonen, 2001; Lozano et al., 2012; Ribas et al., 2011; Wisser et al.,

2005; Witsenboer et al., 1995), suggesting a common mechanism involved in the evolution/action of plant resistance genes. The tendency of resistance genes to cluster seems to occur in all plant species and a model known as ‘the birth and death process’ has been proposed as the origin of the clustering of resistance genes (Michelmore and

Meyers, 1998). The evolution of resistance genes is a dynamic process involving duplication, deletions, sequence exchange, mutations, diversified selection, recombination, gene conversion and retroelement insertion, while the cluster arrangement of resistance genes seems to arise by gene conversion, gene duplication, unequal crossing-over, ectopic recombination or diversifying selection (Friedman and Baker,

2007; Michelmore and Meyers, 1998; Ribas et al., 2011). Just as the taxonomy of R genes implies conserved function, the clustering of these loci implies conserved evolution.

1.3.4. Plant resistance to virus diseases

Plant viruses cause a significant proportion of crop diseases and economic losses around the world (Gomez et al., 2009; Kang et al., 2005). Viruses are obligate intracellular microscopic entities that require host factors for replication and spread. A virus is defined as a nucleoprotein that multiplies only in living cells and has the ability to

12 cause disease (Agrios, 2005). Some can replicate in diverse types of plant cells, while others are limited to the phloem. Viruses have a relatively simple genome with single or double stranded RNA or DNA, and single stranded virus can be positive, negative, or ambi-sense (Redinbaugh and Pratt, 2009). In contrast with other pathogens that cause diseases by consuming or killing host cells with toxins, viruses cause diseases by utilizing the host cellular machinery and disrupting plant cellular process (Agrios,

2005). Most viruses require vectors to spread and move from plant to plant. The vast majority of vectors transmitting viruses are arthropods and a few are transmitted by fungi or nematodes (Agrios, 2005; Lapierre and Signoret, 2004). Vector transmission complicates genetic studies of virus resistance since viral disease establishment requires interactions among virus, viral vector, virus-susceptible germplasm, and environmental conditions (Redinbaugh and Pratt, 2009). Fortunately, relatively straightforward and economical techniques for artificial inoculation in maize are available to facilitate the study of virus diseases and genetic resistance. These methods include vascular puncture inoculation in which the virus is introduced in germinating seeds with the aid of minutes pins attached to an engraving tool (Louie, 1995); rub inoculation, in which the virus is transmitted mechanically by hand rubbing or with the aid of an air brush (Louie et al.,

1983; Louie, 1986); and transmission using insect colonies maintained in the laboratory

(Louie and Anderson, 1993).

Plants have developed genetic mechanisms to suppress virus multiplication and/or spread into other parts of the plant. The use of genetic resistance is considered the most economically and environmentally sustainable approach to control viral disease (Gomez

13 et al., 2009; Redinbaugh and Pratt, 2009). Quantitative and qualitative types of resistance to virus diseases in plants have been reported, but in the vast majority of cases, virus resistance has been conferred by a single gene (Gomez et al., 2009; Kang et al., 2005).

Most of the identified virus resistance genes have dominant inheritance, except for where monogenic recessive resistance is relatively common (Diaz-Pendon et al., 2004). The remaining types of resistance are under polygenic control. Several dominant virus resistance R genes have been isolated from a number of plants, mainly

Arabidopsis and Solanaceous species. Most of these dominant genes encode proteins with

CC, TIR or LZ domains coupled with NBS-LRR domains (Kang et al., 2005).

Nevertheless, RTM1, RTM2, and RTM3 genes for Tobacco etch virus resistance in

Arabidopsis encode a jacalin-like protein, a heat shock protein, and an unknown class of protein, respectively (Chisholm et al., 2001; Cosson et al., 2010). The hypersensitive response (HR) mediated by R genes is similar to that described for other pathogens, but in many cases virus resistance is not associated with HR. (Kang et al., 2005). Maize virus resistance conferred by single dominant genes have been associated with the suppression of systemic virus movement rather than programmed cell death (Redinbaugh and Pratt,

2009).

Several R genes conferring recessive resistance to virus diseases in plants have been described, mainly for viruses in the family Potiviridae (Kang et al., 2005).

Molecular cloning of these recessive genes indicated that mutations in eukaryotic initiation factors, eIF4E and eIF4G, which mediate translation in Arabidopsis were the cause of the resistance (Gururani et al., 2012). It is known that during virus infection

14 eIF4 binds to the VPg region of the virus mimicking the first step of the mRNA translational process (Kang et al., 2005). The most accepted hypothesis that explains recessive resistance to virus diseases is that resistance is the result of the absence of specific factors required by the virus in the host (Diaz-Pendon et al., 2004; Gomez et al.,

2009).

Relatively few quantitative virus resistance genes (QTL) in plants have been studied (Kang et al., 2005). A reason for this is that the analysis of polygenic virus resistance is more complex than monogenic or oligogenic resistance (Gomez et al., 2009) since resistance phenotypes could be transient, the result of virus tolerance (mild symptoms), or highly influenced by gene by gene (GxG) or gene by environment (GxE) interactions (Michelmore, 1995; Poland et al., 2009). A well characterized case of the mechanism of major QTLs conferring partial resistance to Rice yellow mottle virus

(Ioannidou et al., 2003) indicated that partial resistance in rice provided a 1-week delay in virus accumulation in the host tissue. After this time, the virus invaded tissue showing that resistance was transient. The fact that the virus replicated in protoplasts of the resistant cultivar supported the hypothesis that resistance was conferred by a transient lack of movement. In situ hybridization associated the observed partial resistance with delayed virus invasion of the mestome bundle sheaths that can be an efficient barrier to virus movement (Thompson and Garcia-Arenal, 1998). Resistance related to eukaryotic elongation factors seems to be a widespread mechanism of defense against viruses, and any mutation in these regions affects its ability to mediate translation of viral proteins.

15

1.3.5. Mechanisms of virus resistance

Plant virus susceptibility implies that a virus is able to penetrate into the plant cell, replicate in the cell, and move systemically through the whole plant using the plant vascular tissues. Virus infections are usually initiated from contaminated propagules (e.g. seeds, cuttings, bulbs) or vector transmission from a reservoir host (Gomez et al., 2009).

Virus transmission by vectors is a very specialized process that requires the interaction among the virus, vector, and the plant. Vectors transmit virus diseases in different ways when they use their stylets to feed on healthy susceptible plants (Ng and Falk, 2006; Tsai and Falk, 1988). A few plant genes conferring resistance to an insect vector of virus diseases have been cloned and characterized and these belong to the NBS-LRR class of resistance genes (Gomez et al., 2009).

Once the virus enters the cell it must replicate with the aid of the plant cell machinery. Dominant and recessive genes have been identified as responsible for resistance to intracellular virus multiplication (Kang et al., 2005). Examples of dominant genes encoding proteins that inhibit virus replication are tomato Tm-1 and Tm-22. Tm-1 encodes an 80 kDa protein that binds the viral replicase to inhibit replication of Tomato mosaic virus (Ishibashi et al., 2007). The Tm-22 gene encodes a CC-NBS-LRR protein which initiates an HR (Lanfermeijer et al., 2003). Recessive virus resistance genes are mainly identified as alleles of host susceptibility factors belonging to the eIF4E family of translation factors (Gomez et al., 2009; Kang et al., 2005). A well characterized example includes the melon gene nsv controlling resistance to the Carmovirus Melon necrotic spot virus that encodes eIF4E. A single amino acid mutation in this gene is enough to confer

16 resistance (Nieto et al., 2006). Virus resistance controlled by eIF4E has been found in a number of other virus families, including potyviruses, bymoviruses, cucumoviruses, and sobemoviruses (Gomez et al., 2009; Kang et al., 2005). In addition, a few recessive host genes confer resistance to intracellular virus replication, such as Arabidopsis tom1 and tom2A, that confer resistance to Tobacco mosaic virus and encode transmembrane tonoplast proteins that block virus replication (Kang et al., 2005).

After replicating in infected cells, viruses spread to adjacent cells through plasmodesmata, and then to the phloem sieve elements where they are transported over long distances to the rest of the plant. Host susceptibility factors such as eIF4E have been implicated with resistance to cell to cell virus movement, but not with long distance movement (Gomez et al., 2009). The lack of interaction between the eIF4E and virus proteins, including the movement protein (MP), cylindrical inclusion (CI) protein, capside protein (CP), helper component protein (HC-Pro), and the genome-linked protein

(VPg) have shown to be the main cause preventing cell to cell movement (Kang et al.,

2005). Genes encoding NBS-LRR have been also implicated with resistance responses that prevent virus movement from initially-infected leaves. A good example is the

Nicotiana benthamiana N gene, the first virus resistance gene to be cloned, that confers resistance to Tobacco mosaic virus (TMV) in tobacco by induction of an HR. This gene encodes a TIR-NBS-LRR class of protein that is required for viral recognition and triggering of the hypersensitive response (Whitham, 1995). Either recessive resistance related to eukaryotic elongation factors and dominant resistance provided by R genes have been implicated with the suppression of short distance virus movement in plants

17

Relatively little is known about resistance to long distance virus movement because it is more difficult to study (Gomez et al., 2009). A good example of genes conferring resistance to long distance virus movement is found in Arabidopsis resistance to Tobacco etch virus (TEV). The Arabidopsis genes RTM1, RTM2, and RTM3 are responsible for the restriction of the TEV long distance movement, and these genes are expressed in the phloem. RTM1 encodes a protein that belongs to the jacalin repeat family, RTM2 encodes a protein that has similarities to small heat shock proteins, and

RTM3 encodes a protein that belongs to a MEPRIN repeats (Chisholm et al., 2001;

Chisholm et al., 2001; Cosson et al., 2010). The mechanisms by which TEV movement is restricted by these genes is not clear (Cosson et al., 2010; Kang et al., 2005).

RNA silencing, also known as RNAi, is a natural defense mechanism against viruses in plants and other species (Ding and Voinnet, 2007). Genes involved in RNAi are not considered resistance genes. RNAi protects plants from virus diseases by targeting and destroying virus-derived RNA in a sequence-specific manner (Gomez et al.,

2009). In a virus infection, cells have a significant amount of siRNA originating from the virus that is unwound and incorporated into the RNA induced silencing complex (RISC) to target and degrade RNA homologous to the siRNA. The siRNA produced by the plant silencing mechanisms move to adjacent cells more rapidly than the virus allowing the

RISC to immediately recognize and eliminate incoming viruses (Bucher and Prins, 2006), similar to post-transcriptional gene silencing. For example, kohlrabi (Brassica oleracea gongylodes) plants inoculated with Cauliflower mosaic virus (CaMV) showed severe initial symptoms on the inoculated and first systemic leaves, but no virus symptoms or

18 virus accumulation in upper leaves. Additionally, upper leaves were resistance to CaMV and other sequence-related viruses (Covey et al., 1997). Similar effects have been described for Nicotiana clevelandii plants inoculated with Tobacco black ring virus

(Ratcliff et al., 1997). It is suggested that perhaps the role of RNAi in natural resistance is the recovery of infected plants. (Gomez et al., 2009).

1.3.6. Genetics of virus resistance in maize

Despite progress understanding the molecular basis of virus resistance in plants, no virus resistance gene from maize or other grasses have been cloned (Redinbaugh and

Pratt, 2009). Depending on the virus, characterization of the genetic basis of virus resistance in maize has had relatively modest success. Researchers usually face large uncontrolled effects due to high fluctuations in disease pressure and the inter-specific virus-vector-host relationship. Despite these problems, virus resistance genes and QTLs have been identified in maize (McMullen and Simcox 1995; Redinbaugh 2004;

Redinbaugh and Pratt 2009). Characterized virus resistance in maize is primarily dominant and monogenic or oligogenic, such as resistance to the potyviruses MDMV,

SCMV, or WSMV (Ding et al., 2012; McMullen and Louie, 1989;

McMullen and Louie, 1991; McMullen et al., 1994), but it can also be polygenic or quantitative as resistance to MCDV or MMV (Jones et al., 2004; Kyetere et al., 1999;

Rufener et al., 1996). The study of classical Mendelian segregation ratios and QTL analysis provided insights into type of resistance, the mode of action, and the genetic location; however, the number of genes involved in resistance and their mode of action

19 has varied across germplasm and experiments (Jones et al., 2007; Loesch and Zuber,

1967; Louie, 1986; Pokorny and Porubova, 2006; Roane et al., 1983), complicating the analysis and interpretation of the results. This variation has been attributed to the use of diverse maize genetic sources, virus isolates or strains, different classification systems for resistant and susceptible plants, and the presence of genes that modify the activity of resistance loci (Jones et al., 2007; Jones et al., 2011; Louie, 1986), as well as to the presence of disease escapes and environmental effects. Recessive resistance or resistance associated with susceptibility factors has not been identified in maize.

Several virus families have caused significant yield reduction in maize during the last 20 years, including the Potiviridae, , Rhabdoviridae, Tombusviridae,

Tymoviridae, , Sequiviridae, and Tenuiviridae (Redinbaugh and Pratt,

2009). Below I review several families of virus with a focus on those studied in this research.

1.3.6.1. Potyviridae (MDMV, SCMV, and WSMV)

Viruses in the Potyviridae family are considered the most agronomically destructive. They are distributed worldwide in maize and other crops (Ali and Yan, 2012;

Shulka et al., 1994). Viruses in the genus are single-strand positive sense RNA viruses with flexuous rod-shaped virions of about 12 x 750 nm (Lapierre and Signoret,

2004). MDMV and SCMV are the most important potyviruses causing maize dwarf mosaic around the world. MDMV is prevalent in North America and Europe, and SCMV

(formerly known as MDMV-B) is found worldwide (Ali and Yan, 2012; Gordon et al.,

20

1981; Gordon and Thottappilly, 2003; Lapierre and Signoret, 2004). Potyviruses are naturally transmitted in a non-persistent manner by (Shulka et al., 1994), and through seeds at very low rates (<0.5%) (Lapierre and Signoret, 2004). WSMV (genus

Tritimovirus) is transmitted by eriophyid mites, and infects a few maize inbred lines

(LaPierre and Signoret, 2004). WSMV naturally occurs in North America, the Mideast, and Europe, where it mainly affects wheat. WSMV has been model for studies of virus resistance in maize (Redinbaugh and Pratt, 2009).

Genetic resistance to MDMV, SCMV, and WSMV is the best characterized of all virus diseases of maize. Several studies that involved different strains and species of virus, maize populations, environments, and screening techniques have consistently identified resistance genes in specific regions of chromosomes 3, 6 and 10 (Ding et al.,

2012; Dussle et al., 2000; Jones et al., 2004; Jones et al., 2007; McMullen et al., 1994;

Ming et al., 1997; Prazeres De Souza et al., 2008; Redinbaugh and Pratt, 2009; Zhang et al., 2003).

1.3.6.2. Rhabdoviridae (MMV and MFSV)

Maize mosaic disease caused by MMV is common in tropical and subtropical regions of Africa, the Americas, and Hawaii where its main vector Peregrinus maidis is present and sequential overlapping maize crops are grown (Brewbaker, 1981; Lapierre and Signoret, 2004; Ming et al., 1997). Rhabdoviruses have a single-stranded negative sense RNA genome encased in a bullet-shaped virion (Lapierre and Signoret, 2004).

Typical MMV symptoms include fine yellow stripes along leaf veins but not mosaic

21 symptoms, despite the name. Stunting and yield losses are common (Brewbaker, 1981).

MMV is transmitted in a persistent propagative manner by the maize planthopper P. maidis (Redinbaugh and Pratt, 2009). The virus is probably not seed transmitted

(Lapierre and Signoret, 2004).

MFSV has been found only in the United States and the only known vector is the black-faced , Graminella nigrifrons (Todd et al., 2010). Typical disease symptoms include fine chlorotic streaks along the major leaf veins (Redinbaugh et al.,

2002), similar to symptoms associated with MMV. MFSV is considered a model for the study of rhabdovirus-vector, and plant-vector relationships (Chen et al., 2012; Tsai et al.,

2005).

Genetic resistance controlling MMV is relatively well distributed in affected areas. A single QTL conferring resistance to MMV has been mapped on maize chromosome 3 (Ming et al., 1997), in the same region where resistance to MDMV,

SCMV, and WSMV has been reported. To the best of my knowledge, this is the only report about mapping of genetic resistance to MMV. Nothing is known about the genetics of maize resistance to MFSV.

1.3.6.3. Tymoviridae (MRFV)

MRFV is the only member of the genus Marafiviridae known to cause disease in maize. MRFV causes one of the most important maize diseases in some regions of

Mexico, Central and South America (Gamez and Saavedra, 1986; Kogel et al., 1996;

Toler et al., 1985). Severe yield losses, varying from 10% to 83% in maize landraces and

22 nearly 100% in some modern cultivars, have been reported from South and Central

America (Ramirez Rojas et al., 1988; Vandeplas, 2003; Vasquez and Mora, 2007).

MRFV was first reported in Costa Rica in the 1960s (Gamez, 1969). Since then, MRFV has been detected in all Central American countries, Venezuela, Colombia, Ecuador,

Peru, Bolivia, Brazil, Uruguay, , and the southern part of the United States

(Chicas et al., 2007; De-Oliveira et al., 2004; Kogel et al., 1996; Toler et al., 1985). The

MRFV genome is a monopartite single-stranded RNA, and virus particles are symmetrically polyhedral of approximately 30 nm in diameter (Lapierre and Signoret,

2004). In nature, MRFV is persistently transmitted by Dalbulus maidis, which is also an efficient vector for other corn pathogens, such as corn stunt spiroplasma (CSS) and maize bushy stunt phytoplasma (MBSP). Together, these three pathogens form an aggressive disease complex known as “achaparramiento” or red stunt (Nault and Bradfute, 1979).

The susceptibility of maize to MRFV is wide spread. Typical disease symptoms appear on young leaves as small chlorotic spots that become elongated and more numerous along leaf veins with age. Infected plants are reduced in size, which contributes to reduction in grain yield (Toler et al., 1985; Ramirez et al., 1998; Vasquez and Mora,

2007). Most representative maize landraces from the Americas and several hundred hybrids and inbreds were susceptible to the virus (Gamez and Leon, 1985; Valdez et al.,

2004). Toler et al. (1985) evaluated the response of 58 widely commercialized temperate maize inbred lines to MRFV, and no resistant genotypes were found. More than ten years later, Bustamante et al. (1998) evaluated the reaction of 20 tropical maize cultivars to

MRFV. Just two landrace open-pollinated populations, one from the Ecuadorian

23 highlands and the other from the Virgin Islands, showed mild symptoms and low MRFV titer in leaves. Several experiments to identify resistance to MRFV were conducted at the

International Wheat and Maize Improvement Center in Mexico (Ramirez et al., 1998;

Vandeplas 2003). More recently, 68 elite maize genotypes were evaluated, and only one inbred line (CML459) was identified with MRFV resistance under field conditions

(Vandeplas, 2003). Based on wide spread testing of maize accessions, inbreds, and populations, it appears that resistance is rare. Maize populations from the Caribbean and

Andean South America appear the most promising for future screening. Nothing is known about the genetics of maize resistance to MRFV.

1.3.6.4. Secoviridae (MCDV)

Maize chlorotic dwarf is caused by MCDV (genus ) with a positive sense RNA genome and a ca. 30 nm in diameter icosahedral virion (Lapierre and

Signoret, 2004). The disease is distributed from the southeastern U.S. west to Texas and north to Ohio and the Corn Belt (Stewart, 2011). MCDV can cause diverse symptoms including plant stunting, shortening of upper internodes, leaf reddening or yellowing, leaf twisting and tearing, and chlorosis or clearing of the smallest visible leaf veins (vein banding) (Louie and Knoke, 1981). MCDV is transmitted by the leafhopper G. nigrifrons in a semi-persistent manner and there is no evidence for seed transmission (Redinbaugh and Pratt, 2009).

Maize resistance to MCDV is quantitative (Redinbaugh and Pratt, 2009). QTLs conferring additive resistance to MCDV have been mapped on maize chromosomes, 1, 3,

24

5, 7, and 10 (Rufener et al., 1996), and on chromosomes 3, 4, 6, and 10 (Jones et al.,

2004). These QTLs were mapped on F2 populations using the same inbred line Va35 as the susceptible parent and same virus strain. Rufener et al. (1996) used the inbred line

Mp705 as source of resistance and a single inoculation, while Jones et al. (2004) used the inbred line Oh1VI and multiple inoculations. Despite of differences in the source of resistance and methods, QTLs on chromosomes 3 and 10 were common.

1.3.6.5. Tombusviridae (MNeSV)

A virus disease outbreak occurred in 1998 near Cochise, Arizona. plants, thought to be infected with MCDV, were sent to The Ohio State University to be analyzed. In 1 of the 20 samples analyzed, a new maize infecting virus was identified.

This virus was named Maize necrotic streak virus based on the typical symptoms it causes. Virus symptoms on susceptible maize include pale green, yellow, or cream- colored spots and streaks measuring 1 to 2 mm on emerging leaves. Subsequent leaves develop chlorotic bands that later fuse together and become translucent and necrotic around the edges (Louie et al., 2000). MNeSV has a positive sense single-stranded RNA and virions are isometric of approximately 32 nm in diameter (Lapierre and Signoret,

2004). The disease has not been reported elsewhere. The mechanism of MNeSV dispersion in the field under natural conditions is still unknown. Genetic resistance for

MNeSV in maize has been identified (Louie et al., 2000), but nothing is known about the genes conferring resistance to this disease or their mode of inheritance.

25

1.3.7. Clustering of viral resistance genes in maize

The genetic location of loci that confer resistance to virus diseases in maize seems to cluster in specific regions of the genome (Redinbaugh and Pratt, 2009; Redinbaugh et al., 2004; Wisser et al., 2006). Msv1, which confers resistance to

(MSV), and a QTL that confers resistance to Mal del rio cuarto virus (MRCV) have been identified on the short arm of chromosome 1 (Bonamico et al., 2012; Di Renzo et al.,

2004; Kyetere et al., 1999; Welz et al., 1998). Perhaps the most interesting cluster due to the number of virus resistance genes that co- localize is located between bins 3.05 and

3.06. Bins locations are estimated from the B73 v2 reference genome found in Maize

Genetics and Genomic Database (http://www.maizegdb.org/cgi-bin). This region contains

Wsm2 and Scm2 (also known as Scmv2 or Rscmv2) genes that confer resistance to

WSMV. The same region enhances resistance to MDMV, SCMV, and other potyviruses

(Ding et al., 2012; Dussle et al., 2000; Ingvardsen et al., 2010; Jones et al., 2011;

McMullen et al., 1994; Prazeres De Souza et al., 2008; Stewart et al., 2012; Zhang et al.,

2003). The region also contains QTLs that confer resistance to MMV, MCDV, Maize stripe virus (MSpV), and MSV (Dintinger et al., 2005; Jones et al., 2004; Kyetere et al.,

1999; Marcon et al., 1999; Ming et al., 1997; Rufener et al., 1996; Welz et al., 1998).

Another interesting region where clustering has been observed is located on the short arm of chromosome 6, where the major dominant gene, Mdm1, that confers resistance to viruses in the Potyviridae (Jones et al., 2007; Jones et al., 2011; McMullen and Louie,

1989; Stewart et al., 2012) co-localizes with Scmv1 (Xia et al., 1999), Wsm1 (McMullen et al., 1994; Xia et al., 1999), and QTLs conferring resistance to WMoV and Maize rough

26 dwarf virus (MRDV) (Marcon et al., 1999; McMullen et al., 1994; Wang et al., 2007a;

Xia et al., 1999). Yet another clustering of virus resistance genes and QTLs in the maize genome is located between bins 10.05 and 10.06 of the B73 v2 reference genome

(http://www.maizegdb.org/cgi-bin). In this region, Wsm3, which confers resistance to

WSMV (McMullen et al., 1994), co-localizes with QTLs that confer resistance to

MCDV, MSV, and MSpV, and an enhancer of MDMV resistance conferred by Mdm1

(Dintinger et al., 2005; Jones et al., 2004; Pernet et al., 1999a; Pernet et al., 1999b). The four clusters of virus resistance genes also contain loci for bacterial and fungal resistance

(Redinbaugh and Pratt, 2009), as well as putative R genes (Li et al., 2010; Xiao et al.,

2007). The clustering of virus resistance genes and QTLs in the maize genome suggests that the origin and evolution of virus resistance in maize follows the generally accepted model known as ‘the birth and death process’ proposed for all R resistance genes

(Michelmore and Meyers, 1998). In this model new genes are created by gene duplication, whereas others are deleted or become nonfunctional through deleterious mutations (Nei and Rooney, 2005). It is also known that genetic recombination and unqueal crossover between alleles or gene family members frequently create novel variation to change copy number or to create new alleles. (Hulbert et al., 2001). In summary, the cluster of genes in the genome is a complex phenomenon conserved in plant and animals that is driven by selection on allelic variation created by mutations, duplications and recombination between alleles and sometimes different gene families.

27

1.3.8. Breeding for virus resistance in maize

Traditional approaches to breed for virus resistance in plants include: (i) the screening of germplasm collections to identify sources of resistance; (ii) the determination of the mode of inheritance of the resistance and the identification of molecular markers linked to the resistance; and, (iii) the introgression of the resistance into elite lines using marker assisted selection and the evaluation of the performance of the new cultivars under pathogen attack (Gomez et al., 2009). The utilization of disease nurseries in hot spots (places where the disease is severe and commonly found) is the easiest approach to screen for virus resistance in maize. However, this method does not ensure gain under selection because the best way to screen for resistance is through the utilization of appropriate inoculation techniques either in the field or greenhouse

(Redinbaugh and Pratt, 2009). A number of tools used to inoculate viruses in maize are available, including rub inoculation, spray inoculation, insect inoculation, and vascular puncture inoculation (Louie et al., 1983; Louie, 1986; Louie and Anderson, 1993; Louie,

1995).

In maize, most of the disease resistance currently used by breeders is quantitative

(Balint-Kurti and Johal, 2009). The use of backcrosses to transfer the virus resistance

QTLs to elite lines has been commonly used in public and private maize breeding.

Brewbaker et al. (2007) incorporated Mv1, a QTL responsible for resistance to MMV, into 27 virus susceptible inbred lines using at least five cycles of backcrossing and marker assisted selection. Several cycles were needed in order to assure the presence of homozygous MMV resistance alleles and the agronomic attributes of the recurrent lines.

28

Martin et al. (2009) reported successful introgression assisted by markers of a QTL conferring resistance to Mal del rio cuarto virus (MRCV) into elite maize lines. The use of Mdm1 is the only known case of utilization of monogenic virus resistance in maize.

Mdm1 confers complete or partial resistance to MDMV and SCMV (depending on the source of resistance and virus isolate) and it has been used by breeders to develop lines and commercial hybrids with resistance to these diseases (Williams and Pataky, 2012).

To have strong complete resistance, breeders would want to pyramid or combine multiple resistance genes whenever they are available (e.g. Wsm2, Wsm3) since incomplete or partial resistance plants could serve as a reservoir for the virus (Ioannidou et al., 2003;

Redinbaugh and Pratt, 2009).

Molecular breeding has been used to incorporate resistance into maize. Pathogen derived resistance (based on RNAi) has been achieved by expressing viral proteins or

RNAs in maize transgenic plants (Redinbaugh and Pratt, 2009). Transgenic virus resistance lines expressing the coat protein of SCMV, MRFV, MCDV, and MSV have been developed in several parts of the world (McMullen et al., 1996; Shepherd et al.,

2007; Valdez et al., 2004; Zhang et al., 2010). However, the deployment and agronomic utility of these transgenic plants is not known since effective and naturally occurring resistance is available. Further, there may be political and environmental constrains related to the use of a genetically modified organism deployed resistance (Redinbaugh and Pratt, 2009).

29

1.4. Overview of thesis

This dissertation contains six chapters, including this literature review. Chapter 1 introduces the importance of the research and its objectives. Additionally, it provides information about the current state of knowledge of virus resistance in plants in general and maize in specific. Chapter 2 “Identification of resistance to Maize rayado fino virus in maize inbred lines” has been accepted for publication in Plant Disease. This chapter describes novel sources of resistance to MRFV and the mode of inheritance observed in the F1 and F2 generations of one of the identified resistant lines. Chapter 3 “Genetic analysis of resistance to six virus diseases in a multiple virus-resistant maize inbred line” has been submitted to Theoretical and Applied Genetics. It describes the analysis of

Mendelian segregation ratios of the resistance to MDMV, SCMV, MFSV, MMV, and

MCDV in F1 and F2 generations, and the mapping of the genes or QTLs conferring resistance to these diseases in a recombinant inbred line (RIL) population. Chapter 4

“QTL mapping of resistance to Maize rayado fino virus” describes the first mapping of

QTL conferring resistance to MRFV in two RIL populations. The manuscript is in preparation for submission to Molecular Breeding. Chapter 5 “Identification of a major quantitative trait locus controlling resistance to Maize necrotic streak virus in maize” reports the first mapping of a QTL conferring resistance to MNeSV in two RIL populations and the mode of inheritance of the resistance observed in their F1 and F2 generations. The manuscript is in preparation for submission to Phytopathology. Chapter

6 “Agronomic evaluation of a recombinant inbred line maize population and selection of multi-virus resistant lines with superior agronomic traits” describes the selection and

30 agronomic evaluation of maize inbred lines that are resistant to multiple virus diseases.

Additionally, it compares the virus resistance responses with the agronomic performance of the RIL population.

The most important contributions of this work are: the identification of novel sources of resistance to MRFV, the first identification of QTLs conferring resistance to

MRFV, MFSV, and MNeSV, the genetic characterization of multi-virus resistance in the maize inbred line Oh1VI, and the identification of clusters of QTLs conferring resistance to phylogenetically diverse viruses.

31

1.5. References

Agrios, G.N. 2005. . Elsevier Academic Press Burlington, MA.

Ali, F., and Yan, J. 2012. Disease Resistance in Maize and the Role of Molecular Breeding in Defending Against Global Threat. Journal of Integrative Plant Biology 54:134-151.

Balint-Kurti, P., and Johal, G.S. 2009. Maize disease resistance. In: Handbook of maize: Its Biology. Bennetzen, J.L., andHake, S.C., eds.Springer, New York. pp 229-250.

Boller, T., and Felix, G. 2009. A Renaissance of Elicitors: Perception of Microbe- Associated Molecular Patterns and Danger Signals by Pattern-Recognition Receptors. Annual Review of Plant Biology 60:379-406.

Bonamico, N.C., Di Renzo, M.A., Ibañez, M.A., Borghi, M.L., Díaz, D.G., Salerno, J.C., and Balzarini, M.G. 2012. QTL analysis of resistance to Mal de Río Cuarto disease in maize using recombinant inbred lines. The Journal of Agricultural Science 150:619-629.

Bonas, U., and Lahaye, T. 2002. Plant disease resistance triggered by pathogen-derived molecules: refined models of specific recognition. Curr Opin Microbiol 5:44-50.

Brewbaker, J.L. 1981. Resistance to Maize mosaic virus. In: Virus and Virus-like Disease of Maize in the United States. 247th ed. Gordon, D.T., Knoke, J.K., and Scott, G.E., eds. Southern Cooperative Series Bulletin, Wooster, Ohio. pp. 145-151.

Bucher, E., and Prins, M. 2006. RNA silencing: a natural resistance mechanism in plants. In: Natural Resistance Mechanism of Plant Viruses. Loebenstein GC and Carr JP, eds. Springer, The Netherlands. pp 45-72.

Bustamante, P.I., Ramirez, P., and Hammond, R. 1998. Evaluation of maize germplasm for resistance to Maize rayado fino virus. Plant Dis 82:50-56.

Castillo, J., and Hebert, T.T. 1974. A new virus disease of maize in Peru. Phytopathology 9:79-84.

Chahal, G.S., and Gosal, S.S. 2002. Principles and procedures of . Alpha Science International Ltd., United Kingdom.

Chen, Y., Cassone, B.J., Bai, X., Redinbaugh, M.G., and Michel, A.P. 2012. Transcriptome of the plant virus vector Graminella nigrifrons, and the molecular interactions of Maize fine streak rhabdovirus transmission. Plos One 7:e40613.

32

Chicas, M., Caviedes, M., Hammond, R., Madriz, K., Albertazzi, F., Villalobos, H., and Ramírez, P. 2007. Partial characterization of Maize rayado fino virus isolates from Ecuador: Phylogenetic analysis supports a Central American origin of the virus. Virus Res 126:268-276.

Chisholm, S.T., Coaker, G., Day, B., and Staskawicz, B.J. 2006. Host-microbe interactions: Shaping the evolution of the plant immune response. Cell 124:803-814.

Chisholm, S.T., Parra, M.A., Anderberg, R.J., and Carrington, J.C. 2001. Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus. Plant Physiol 127:1667-1675.

Cosson, P., Sofer, L., Le, Q.H., Leger, V., Schurdi-Levraud, V., Whitham, S.A., Yamamoto, M.L., Gopalan, S., Le Gall, O., Candresse, T., Carrington, J.C., and Revers, F. 2010. RTM3, which controls long-distance movement of potyviruses, is a member of a new plant gene family encoding a MEPRIN and TRAF homology domain-containing protein. Plant Physiol 154:222-232.

Covey, S.N., AlKaff, N.S., Langara, A., and Turner, D.S. 1997. Plants combat infection by gene silencing. Nature 385:781-782.

Dangl, J.L., and Jones, J.D.G. 2001. Plant pathogens and integrated defence responses to infection. Nature 411:826-833.

De-Oliveira, E., Duarte, A.P., De-Carvalho, R., and De-Oliveira, A.C. 2004. Molicutes e virus na cultura do milho no brasil: caracterizacao e factores que afetam sua incidencia. In: Doencas en Milho. Molicutes, Virus, Vetores e Mancha por Phaeosphaeria. in: De- Oliveira, E., andDe-Oliveira, C.M., eds.Embrapa Informacao Tecnologica, Brasilia pp 17-34.

Di Renzo, M.A., Bonamico, N.C., Diaz, D.G., Ibanez, M.A., Faricelli, M.E., Balzarini, M.G., and Salerno, J.C. 2004. Microsatellite markers linked to QTL for resistance to mal de rio cuarto disease in mays L. J Agric Sci 142:289-295.

Diaz-Pendon, J., Truniger, V., Nieto, C., Garcia-Mas, J., Bendahmane, A., and Aranda, M. 2004. Advances in understanding recessive resistance to plant viruses. Mol Plant Pathol 5:223-233.

Ding, J., Li, H., Wang, Y., Zhao, R., Zhang, X., Chen, J., Xia, Z., and Wu, J. 2012. Fine mapping of Rscmv2, a major gene for resistance to Sugarcane mosaic virus in maize. Mol Breed 30:1593-1600.

Ding, S., and Voinnet, O. 2007. Antiviral immunity directed by small . Cell 130:413-426. 33

Dintinger, J., Verger, D., Caiveau, S., Risterucci, A.M., Gilles, J., Chiroleu, F., Courtois, B., Reynaud, B., and Hamon, P. 2005. Genetic mapping of maize stripe disease resistance from the Mascarene source. Theor Appl Genet 111:347-359.

Dussle, C.M., Melchinger, A.E., Kuntze, L., Stork, A., and Luebberstedt, T. 2000. Molecular mapping and gene action of Scm1 and Scm2, two major QTL contributing to SCMV resistance in maize. Plant Breeding 119:299-303.

Flor, H.H. 1971. Current Status of the Gene for Gene Concept. Annu. Rev. of Phytopathology 9:275-296.

Friedman, A.R., and Baker, B.J. 2007. The evolution of resistance genes in multi-protein plant resistance systems. Curr Opin Genet Dev 17:493-499.

Gamez, R. 1969. A new leafhopper-borne virus of corn in Central America. Plant disease reporter 12:929-932.

Gamez, R., and Leon, P. 1985. Ecology and evolution of a Neotropical leafhopper-virus - maize association. In: The and planthoppers. in: Nault, L.R., and Rodriguez, J.G., eds.John Wiley & Sons Inc., New York. pp. 331-349.

Gamez, R., and Saavedra, F. 1986. Maize rayado fino: a model of a leafhopper-borne virus disease in the Neotropics. In: Plant virus epidemics : monitoring, modelling and predicting outbreaks / edited by George D. McLean, Ronald G. Garrett, William G. Ruesink. Sydney : Academic Press, c1986. pp. 315-326.

Gebhardt, C., and Valkonen, J.P.T. 2001. Organization of genes controlling disease resistance in the potato genome. Annu Rev Phytopathol 39:79-102.

Gimenez-Pecci, M., Nome, C.F., Laguna, I.G., Borgogno, C., Oliveira, E., and Resende, R. 2000. Occurrence of Maize rayado fino virus in maize in Argentina. Plant Dis 84:1046-1046.

Gomez, P., Rodriguez-Hernandez, A.M., Moury, B., and Aranda, M.A. 2009. Genetic resistance for the sustainable control of plant virus diseases: breeding, mechanisms and durability. Eur J Plant Pathol 125:1-22.

Gordon, D.T., Bradfute, O.E., Gingery, R.E., Knoke, J.K., Nault, L.R., and Scott, G.E. 1981. Introduction: history, geographical distribution, pathogen characteristics and economic importance. In: Virus and Virus-like Disease of Maize in the United States. 247th ed. in: Gordon, D.T., Knoke, J.K., and Scott, G.E., eds. Southern Cooperative Series Bulletin, Wooster, Ohio. pp. 1-12.

34

Gordon, D.T., and Thottappilly, G. 2003. Maize and Sorghum. In: Virus and virus-like diseases of major crops in developing countries. in: Loebenstein, G., andThottappilly, G., eds.Kluwer Academic Publishers, The Netherlands. pp. 295-334.

Guest, D., and Brown, J.F. 1997. Plant defenses against pathogens. In: Plant pathogens and plant diseases. Brown, J.F., andOgle, H.J., eds.Rockvale Publications, Australia. pp. 263-286.

Gururani, M.A., Venkatesh, J., Upadhyaya, C.P., Nookaraju, A., Pandey, S.K., and Park, S.W. 2012. Plant disease resistance genes: Current status and future directions. Physiol Mol Plant Pathol 78:51-65.

Hamilton, J.P., and Buell, C.R. 2012. Advances in plant genome sequencing. Plant Journal 70:177-190.

Hulbert, S., Webb, C., Smith, S., and Sun, Q. 2001. Resistance gene complexes: Evolution and utilization. Annu Rev Phytopathol. 39:285-312.

Ingvardsen, C.R., Xing, Y., Frei, U.K., and Luebberstedt, T. 2010. Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize. Theor Appl Genet 120:1621-1634.

Ioannidou, D., Pinel, A., Brugidou, C., Albar, L., Ahmadi, N., Ghesquiere, A., Nicole, M., and Fargette, D. 2003. Characterization of the effects of a major QTL of the partial resistance to Rice yellow mottle virus using a near-isogenic-line approach. Physiol Mol Plant Pathol 63:213-221.

Ishibashi, K., Masuda, K., Naito, S., Meshi, T., and Ishikawa, M. 2007. An inhibitor of viral RNA replication is encoded by a plant resistance gene. Proc Natl Acad Sci U S A 104:13833-13838.

Johal, D.S., and Briggs, S.P. 1992. Reductase activity encoded by the HM1 disease resistance gene in maize. Science 258:985-987.

Jones, J.D.G., and Dangl, J.L. 2006. The plant immune system. Nature 444:323-329.

Jones, M.W., Redinbaugh, M.G., Anderson, R.J., and Louie, R. 2004. Identification of quantitative trait loci controlling resistance to Maize chlorotic dwarf virus. Theor Appl Genet 110:48-57.

Jones, M.W., Redinbaugh, M.G., and Louie, R. 2007. The Mdm1 locus and maize resistance to Maize dwarf mosaic virus. Plant Dis 91:185-190.

35

Jones, M.W., Boyd, E.C., and Redinbaugh, M.G. 2011. Responses of maize (Zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the Potyviridae. Theor Appl Genet 123:729-740.

Kang, B., Yeam, I., and Jahn, M.M. 2005. Genetics of plant virus resistance. Annu Rev Phytopathol 43:581-621.

Kang, L., Li, J.X., Zhao, T.H., Xiao, F.M., Tang, X.Y., Thilmony, R., He, S.Y., and Zhou, J.M. 2003. Interplay of the Arabidopsis nonhost resistance gene NHO1 with bacterial virulence. Proc Natl Acad Sci 100:3519-3524.

Kanzaki, H., Saitoh, H., Ito, A., Fujisawa, S., Kamoun, S., Katou, S., Yoshioka, H., and Terauchi, R. 2003. Cytosolic HSP90 and HSP70 are essential components of INF1- mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Molecular Plant Pathology 4:383-391.

Kogel, R., Ramirez, P., and Hammond, R.W. 1996. Incidence and geographic distribution of Maize rayado fino virus (MRFV) in Latin America. Plant Dis 80:679-683.

Kump, K.L., Bradbury, P.J., Wisser, R.J., Buckler, E.S., Belcher, A.R., Oropeza-Rosas, M.A., Zwonitzer, J.C., Kresovich, S., McMullen, M.D., Ware, D., Balint-Kurti, P.J., and Holland, J.B. 2011. Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population. Nat Genet 43:161-169.

Kyetere, D.T., Ming, R., McMullen, M.D., Pratt, R.C., Brewbaker, J., and Musket, T. 1999. Genetic analysis of tolerance to maize streak virus in maize. Genome 42 1:20-26.

Lanfermeijer, F.C., Dijkhuis, J., Sturre, M.J.G., de Haan, P., and Hille, J. 2003. Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-2(2) from Lycopersicon esculentum. Plant Mol Biol 52:1037-1049.

Lapierre, H., and Signoret, P.A. 2004. Viruses and virus diseases of (Gramineae). INRA. Paris, France.

Li, J., Ding, J., Zhang, W., Zhang, Y., Tang, P., Chen, J., Tian, D., and Yang, S. 2010. Unique evolutionary pattern of numbers of gramineous NBS-LRR genes. Molecular Genetics and Genomics 283:427-438.

Loesch, P.J., and Zuber, M.S. 1967. An Inheritance Study of Resistance to Maize dwarf mosaic virus in corn (Zea Mays L). Agron J 59:423-&.

Louie, R., and Knoke, J.K. 1981. Symptoms and disease diagnostic. In: Virus and Virus- like Disease of Maize in the United States. 247th ed. in: Gordon, D.T., Knoke, J.K., andScott, G.E., eds.Southern Cooperative Series Bulletin, Wooster, Ohio.pp. 13-18. 36

Louie, R., Knoke, J.K., and Reichard, D.L. 1983. Transmission of Maize dwarf mosaic virus with solid-stream inoculum. Plant Dis 67:1328-1331.

Louie, R. 1986. Effects of Genotype and inoculation protocols on resistance evaluation of maize to Maize dwarf mosaic virus strains. Phytopathology 76 8:769-773.

Louie, R., and Anderson, R.J. 1993. Evaluation of Maize chlorotic dwarf virus resistance in maize with multiple inoculations by Graminella nigrifrons (Homoptera: Cicadellidae). J Econ Entomol 86:1579-1583.

Louie, R. 1995. Vascular puncture of maize kernels for the mechanical transmission of Maize white line mosaic virus and other viruses of maize. Phytopathology 85:139-143.

Louie, R., Abt, J.J., Anderson, R.J., Redinbaugh, M.G., and Gordon, D.T. 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family Tombusviridae. Plant Dis 84:1133-1139.

Lozano, R., Ponce, O., Ramirez, M., Mostajo, N., and Orjeda, G. 2012. Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja. Plos One 7:e34775.

Mansfield, J.W. 2009. From bacterial avirulence genes to effector functions via the hrp delivery system: an overview of 25 years of progress in our understanding of plant innate immunity. Molecular Plant Pathology 10:721-734.

Marcon, A., Kaeppler, S.M., Jensen, S.G., Senior, L., and Stuber, C. 1999. Loci controlling resistance to High plains virus and Wheat streak mosaic virus in a B73 x Mo17 population of maize. Crop Sci 39:1171-1177.

Martin, T., Franchino, J.A., Kreff, E.D., Procopiuk, A.M., Tomas, A., Luck, S.D., and Shu, G.G. 2009. Major QTL conferring resistance of corn to . Patent US2008/012327.

McDowell, J.M., and Woffenden, B.J. 2003. Plant disease resistance genes: recent insights and potential applications. Trends Biotechnol 21:178-183.

McMullen, M.D., and Louie, R. 1989. The linkage of molecular markers to a gene controlling the symptom response in maize to Maize dwarf mosaic virus. Mol Plant- Microbe Interact 2:309-314.

McMullen, M.D., and Louie, R. 1991. Identification of a gene for resistance to Wheat streak mosaic virus in maize. Phytopathology 81:624-627.

37

McMullen, M.D., Louie, R., Simcox, K.D., and Jones, M.W. 1994. Three genetic loci control resistance to Wheat streak mosaic virus in the maize inbred Pa405. Molecular plant-microbe interactions 7:708-712.

McMullen, M.D., Roth, B.A., and Townsend, R. 1996. Maize chlorotic dwarf virus and resistance thereto. Official Gazette of the United States Patent and Trademark Office Patents 1191 5:3527-3527.

Michelmore, R. 1995. Molecular approaches to manipulation of disease resistance genes. Annu Rev Phytopathol 33:393-427.

Michelmore, R.W., and Meyers, B.C. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res 8:1113-1130.

Ming, R., Brewbaker, J.L., Pratt, R.C., Musket, T.A., and McMullen, M.D. 1997. Molecular mapping of a major gene conferring resistance to Maize mosaic virus. Theor Appl Genet 95:271-275.

Mysore, K.S., and Ryu, C.M. 2004. Nonhost resistance: how much do we know? Trends Plant Sci 9:97-104.

Nault, L.R., and Bradfute, O.E. 1979. Corn stunt: involvement of a complex of leafhopper-borne pathogens. In: Leafhopper vectors and plant disease agents. in: Maramorosch, K., andHarris, K.F., eds.Academic Press, New York. pp. 561-586.

Nei, M., and Rooney, A. 2005. Concerted and birth-and-death evolution of multigene families. Annu Rev Genet 39:121-152.

Ng, J.C.K., and Falk, B.W. 2006. Virus-vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annu Rev Phytopathol 44:183-212.

Nieto, C., Morales, M., Orjeda, G., Clepet, C., Monfort, A., Sturbois, B., Puigdomenech, P., Pitrat, M., Caboche, M., Dogimont, C., Garcia-Mas, J., Aranda, M.A., and Bendahmane, A. 2006. An eIF4E allele confers resistance to an uncapped and non- polyadenylated RNA virus in melon. Plant Journal 48:452-462.

Pernet, A., Hoisington, D., Dintinger, J., Jewell, D., Jiang, C., Khairallah, M., Letourmy, P., Marchand, J.L., Glaszmann, J.C., and de Leon, D.G. 1999. Genetic mapping of Maize streak virus resistance from the Mascarene source. II. Resistance in line CIRAD390 and stability across germplasm. Theor Appl Genet 99:540-553.

Pernet, A., Hoisington, D., Franco, J., Isnard, M., Jewell, D., Jiang, C., Marchand, J.L., Reynaud, B., Glaszmann, J.C., and de Leon, D.G. 1999. Genetic mapping of Maize streak

38 virus resistance from the Mascarene source. I. Resistance in line D211 and stability against different virus clones. Theor Appl Genet 99:524-539.

Pokorny, R., and Porubova, M. 2006. Heritability of resistance in maize to the Czech isolate of Sugarcane mosaic virus. Research Communications 34:1081-1086.

Poland, J.A., Pratt, R.C., Nelson, R.J., Balint-Kurti, P., and Wisser, R.J. 2009. Shades of gray: the world of quantitative disease resistance. Trends Plant Sci 14:21-29.

Poland, J.A., Bradbury, P.J., Buckler, E.S., and Nelson, R.J. 2011. Genome-wide nested association mapping of quantitative resistance to northern leaf blight in maize. Proc Natl Acad Sci. 108:6893-6898.

Prazeres De Souza, I.R., Schuelter, A.R., Guimaraes, C.T., Schuster, I., De Oliveira, E., and Redinbaugh, M. 2008. Mapping QTL contributing to SCMV resistance in tropical maize. Hereditas (Lund):167-173.

Ramirez Rojas, S., Romero Rosales, F., Dan, J., Martinez Garza, A., and Mejia Andrade, H. 1988. Reacción de ocho variedades de maíz al virus del rayado fino en Chapingo, México. Agricultura Tecnica en Mexico 24:11-18.

Ratcliff, F., Harrison, B.D., and Baulcombe, D.C. 1997. A similarity between viral defense and gene silencing in plants. Science 276:1558-1560.

Redinbaugh, M.G., Houghton, W., Salomon, R., Creamer, R., Hogenhout, S.A., Gordon, D.T., Ackerman, J., Meulia, T., Seifers, D.L., Abt, J.J., Styer, W.E., and Anderson, R.J. 2002. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathology 92:1167-1174.

Redinbaugh, M.G., Gingery, R.E., and Jones, M.W. 2004. The genetics of virus resistance in maize (Zea mays L.). Maydica 49:183-190.

Redinbaugh, M.G., and Pratt, R.C. 2009. Virus Resistance. In: Handbook of maize: Its Biology. in: Bennetzen, J.L., andHake, S.C., eds.Springer, New York. pp. 251-268.

Ribas, A.F., Cenci, A., Combes, M., Etienne, H., and Lashermes, P. 2011. Organization and molecular evolution of a disease-resistance gene cluster in coffee trees. BMC Genomics 12:240.

Roane, C.W., Genter, C.F., and Tolin, S.A. 1983. Inheritance of resistance to Maize dwarf mosaic virus in maize inbred line Oh7B. Phytopathology 73 6:845-850.

39

Rufener, G.K., Balducchi, A.J., Mowers, R.P., Pratt, R.C., Louie, R., McMullen, M.D., and Knoke, J.K. 1996. Maize chlorotic dwarf virus resistant maize and the producction thereof. USA patent 5,563,316.

Shepherd, D.N., Mangwende, T., Martin, D.P., Bezuidenhout, M., Kloppers, F.J., Carolissen, C.H., Monjane, A.L., Rybicki, E.P., and Thomson, J.A. 2007. Maize streak virus-resistant transgenic maize: a first for Africa. Plant Biotechnology Journal 5:759- 767.

Shulka, D.D., Wardand, C.W., and Brunt, A.A. 1994. The Potyviridae. CAB International, Oxon, UK.

Stewart, L.R. 2011. Waikaviruses: studied but not understood. APSnet Features APS Press Verified 2013 05/16.

Stewart, L.R., Md, A.H., Jones, M.W., and Redinbaugh, M.G. 2012. Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus. Mol Breed. 31:289-297.

Thompson, J.R., and Garcia-Arenal, F. 1998. The bundle sheath-phloem interface of Cucumis sativus is a boundary to systemic infection by Tomato aspermy virus. Mol Plant- Microbe Interact 11:109-114.

Todd, J.C., Hoy, C., Hogenhout, S.A., Ammar, E., and Redinbaugh, M.G. 2010. Plant host range and leafhopper transmission of Maize fine streak virus. Phytopathology 100:1138-1145.

Toler, R.W., Harris, K.F., Bockholt, A.J., and Skinner, G. 1985. Reactions of maize (Zea mays) accessions to Maize rayado fino virus. Plant Dis 69:56-57.

Tsai, J.H., and Falk, B.W. 1988. Tropical maize pathogens and their associated insect vector. In: Advances in disease vectors research. in: Harris, K.F., ed.Springer - Verlog, New York. pp. 177-201.

Tsai, C., Goodin, M., Hogenhout, S.A., Reed, S., Redinbaugh, M.G., and Willie, K.J. 2005. Complete genome sequence and in planta subcellular localization of Maize fine streak virus proteins. Journal of virology 79:5304-5314.

Uyemoto, J.K., Bockelman, D.L., and Claflin, L.E. 1980. Severe Outbreak of Corn Lethal Necrosis Disease in Kansas. Plant Dis 64:99-100.

Valdez, M., Madriz, K., and Ramírez, P. 2004. A method for genetic transformation of maize for resistance to viral diseases. Rev Biol Trop 52:787-793.

40 van der Biezen, E.A., and Jones, J.D.G. 1998. Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem Sci 23:454-456. van Eeuwijk, F. Genotype by environment interaction-basics and beyond. In: Plant Breeding: The Arnel R. Hallauer International Symposium. Lamkey and Lee Eds. Blackwell Publishing, Iowa, U.S. pp. 155-170.

Vandeplas A. 2003. Evaluation of sixty highland elite maize genotypes for resistance to Maize rayado fino virus. Dissertation. Belgium: The Katholieke Universiteit Leuven.

Vasquez, J., and Mora, E. 2007. Incidence of and yield loss caused by Maize rayado fino virus in maize cultivars in Ecuador. Euphytica 153:339-342.

Wang, F., Zhang, Y.S., Zhuang, Y.L., Qin, G.Z., and Zhang, J.R. 2007. Molecular mapping of three loci conferring resistance to maize (Zea mays L.) rough dwarf disease. Mol Plant Breed 5:178-179.

Wang, G., Chen, Y., Zhao, J., Li, L., Korban, S.S., Wang, F., Li, J., Dai, J., and Xu, M. 2007. Mapping of defense response gene homologs and their association with resistance loci in maize. Journal of Integrative Plant Biology 49:1580-1598.

Wangai, A.W., Redinbaugh, M.G., Kinyua, Z.M., Miano, D.W., Leley, P.K., Kasina, M., Mahuku, G., Scheets, K., and Jeffers, D. 2012. First report of Maize chlorotic mottle virus and Maize Lethal Necrosis in Kenya. Plant Disease 96:1582-1582.

Welz, H.G., Schechert, A., Pernet, A., Pixley, K.V., and Geiger, H.H. 1998. A gene for resistance to the Maize streak virus in the African CIMMYT maize inbred line CML202. Mol Breed 4:147-154.

Whitham. 1995. The Product of the Tobacco mosaic virus resistance gene-N - similarity to Toll and the Interleukin-1 receptor. Cell 81:466-466.

Williams, M.M.,II, and Pataky, J.K. 2012. Interactions between maize dwarf mosaic and weed interference on sweet corn. Field Crops Res 128:48-54.

Wisser, R.J., Sun, Q., Hulbert, S.H., Kresovich, S., and Nelson, R.J. 2005. Identification and characterization of regions of the rice genome associated with broad-spectrum, quantitative disease resistance. Genetics 169:2277-2293.

Wisser, R.J., Nelson, R.J., and Balint-Kurti, P. 2006. The genetic architecture of disease resistance in maize: A synthesis of published studies. Phytopathology 96:120-129.

41

Witsenboer, H., Kesseli, R.V., Fortin, M.G., Stanghellini, M., and Michelmore, R.W. 1995. Sources and genetic-structure of a cluster of genes for resistance to 3 pathogens in lettuce. Theor Appl Genet 91:178-188.

Wu, J., Ding, J., Du, Y., Xu, Y., and Zhang, X. 2007. Genetic analysis and molecular mapping of two dominant complementary genes determining resistance to Sugarcane mosaic virus in maize. Euphytica 156 3:355-364.

Xia, X.C., Melchinger, A.E., Kuntze, L., and Lubberstedt, T. 1999. Quantitative trait loci mapping of resistance to Sugarcane mosaic virus in maize. Phytopathology 89:660-667.

Xiao, W., Zhao, J., Fan, S., Li, L., Dai, J., and Xu, M. 2007. Mapping of genome-wide resistance gene analogs (RGAs) in maize (Zea mays L.). Theor Appl Genet 115:501-508.

Young, N.D. 1996. QTL mapping and quantitative disease resistance in plants. Annu Rev Phytopathol 34:479-501.

Zhang, S.H., Li, X.H., Wang, Z.H., George, M.L., Jeffers, D., Wang, F.G., Liu, X.D., Li, M.S., and Yuan, L.X. 2003. QTL mapping for resistance to SCMV in Chinese maize germplasm . Maydica 48:307-312.

Zhang, S.Q., and Klessig, D.F. 2001. MAPK cascades in plant defense signaling. Trends Plant Sci 6:520-527.

Zhang, Z., Fu, F., Gou, L., Wang, H., and Li, W. 2010. RNA interference-based transgenic maize resistant to Maize dwarf mosaic virus. Journal of Plant Biology 53:297- 305.

Zwonitzer, J.C., McMullen, M.D., Pratt, R.C., Balint-Kurti, P., Holland, J.B., Coles, N.D., Krakowsky, M.D., and Arellano, C. 2010. Mapping resistance quantitative trait loci for three foliar diseases in a maize recombinant inbred line population-evidence for multiple disease resistance? Phytopathology 100:72-79.

42

CHAPTER 2

IDENTIFICATION OF RESISTANCE TO MAIZE RAYADO FINO VIRUS IN

MAIZE INBRED LINES

2.1. Abstract

Maize rayado fino virus (MRFV) causes one of the most important virus diseases of maize in America. Severe yield losses, ranging from 10 to 50% in landraces to nearly

100% in contemporary cultivars, have been reported. Resistance has been reported in maize populations, but few resistant inbred lines have been identified. Maize inbred lines representing the range of diversity in the cultivated types and selected lines known to be resistant to other viruses were evaluated to identify novel sources of resistance to MRFV.

The virus was transmitted to maize seedlings using the vector Dalbulus maidis, and disease incidence and severity were evaluated beginning seven days post inoculation.

Most of the 36 lines tested were susceptible to MRFV, with mean disease incidence ranging from 21 to 96%, and severity from 1.0 to 4.3 (using a 0 to 5 severity scale). A few genotypes, including CML333 and Ki11, showed intermediate levels of resistance, with 14% and 10% incidence, respectively. Novel sources of resistance, with incidence of less than 5% and severity ratings of 0.4 or less, included the inbred lines Oh1VI,

43

CML287, and Cuba. In Oh1VI, resistance appeared to be dominant, and segregation of resistance in F2 plants was consistent with one or two resistance genes. The discovery of novel sources of resistance in maize inbred lines will facilitate the identification of virus resistance genes and their incorporation into breeding programs.

2.2. Introduction

Maize rayado fino virus (MRFV) is one of the most important virus diseases of maize (Zea mays L.) in parts of Mexico, and Central and South America (De-Oliveira et al., 2004; Gordon and Thottappilly, 2003; Kogel et al., 1996; Redinbaugh and Pratt,

2009). Severe yield losses, ranging from 10 to 50% in landrace populations to nearly

100% in some cultivars, have been reported (Gamez, 1983; Valdez et al., 2004; Vasquez and Mora, 2007). First described in El Salvador (Gamez, 1969), the disease is often found in plants co-infected with corn stunt spiroplasma (CSS; Spiroplasma kunkelii) and maize bushy stunt phytoplasma (MBSP; Candidatus Phytoplasma astris subgroup 16SrI-B).

Together, the three pathogens form an aggressive disease complex known as

“achaparramiento” or red stunt (Nault and Bradfute, 1979).

MRFV is the type member of the genus . Virus particles are icosahedra approximately 30 nm in diameter (Gamez and Leon, 1988). The virus genome is a 6305 nucleotide monopartite, single stranded, positive sense RNA (Hammond and

Ramirez, 2001). Genetic variation among MRFV populations has been described, with four groups or races occurring in the Americas (Chicas et al., 2007). In nature, MRFV is transmitted by the corn leafhopper Dalbulus maidis (Homoptera: Cicadellidae) and it

44 replicates in both its host plant and insect vector (Gamez and Leon, 1985). Nymphs and adults can transmit MRFV to plants, but the virus is not transmitted transovarially. The latent period between virus acquisition and subsequent transmission by vectors is temperature dependent, and varies from 8 to 37 days. Under experimental conditions,

MRFV can be also transmitted by the leafhoppers D. elimatus, Stirellus bicolor and

Graminella nigrifrons. The disease is not transmitted by seed (Nault et al., 1980). Typical disease symptoms appear on young leaves as small chlorotic spots that become elongated and more numerous along leaf veins with age (Gamez and Leon, 1988; Toler et al.,

1985). Susceptible plants infected at early developmental stages are reduced in height and develop ears with few or no seed, resulting in yield reduction (Gordon and Thottappilly,

2003; Vasquez and Mora, 2007).

There have been several attempts to identify sources of genetic resistance to

MRFV since the identification of the virus in Central America (Gamez, 1969). Several hundred cultivars, from landraces to inbred lines, have been reported as susceptible to

MRFV infection, but only three tropical accessions, Saint Croix, INIAP176 and

CML459, were identified as resistant (Bustamante et al., 1998; Espinoza and Gamez,

1980; Ramirez-Rojas et al., 1998; Toler et al., 1985; Vandeplas, 2003). Saint Croix and

INIAP176 are open pollinated cultivars, complicating the use of this germplasm for genetic analysis. Although CML459 is a highland inbred line, no mapping populations using this line are available. The objective of this study was to identify novel sources of resistance to MRFV in a set of tropical and temperate inbred lines that will support further genetic analysis and that may be directly used in breeding programs.

45

2.3. Materials and methods

2.3.1. Maize germplasm

Seeds of 36 maize accessions were obtained from the North Central Regional

Plant Introduction Station (NCRPIS) and the International Maize and Wheat

Improvement Center (CIMMYT), or were maintained by the USDA, ARS Corn, Soybean and Wheat Quality Research Unit (CSWQRU) in Wooster, OH as indicated in Table 2.1.

Accessions were selected based on the availability of mapping populations and/or resistance to other virus diseases, with most of the selected genotypes corresponding to the founders of the Nested Association Mapping (NAM) population (McMullen et al.,

2009; Yu et al., 2008). The susceptible genotypes ‘Spirit’ (Syngenta, Idaho) and B73, and the resistant cultivars Saint Croix (PI484036), INIAP180 (derived from INIAP-176), and

CML459 were used as controls.

2.3.2. Virus isolate and insect vector

The sweet corn hybrid ‘Spirit’ was used for virus maintenance. A MRFV isolate collected in Texas by Bradfute and others (Bradfute et al., 1979) was used. The isolate was stored in liquid nitrogen, then transmitted to germinating maize kernels using vascular puncture inoculation (Louie, 1995; Madriz-Ordenana et al., 2000), and subsequently maintained by serial transmission to maize seedlings using a D. maidis colony originally collected from California (Nault et al., 1980). Viruliferous D. maidis were obtained by allowing adults reared on healthy maize to feed on MRFV-infected

46 plants for 26 days before being used for inoculation (Bustamante et al., 1998; Toler et al.,

1985).

2.3.3. Evaluation of maize responses to MRFV

Single seeds of each accession were planted into 16.4 x 2.5 cm “Cone-tainers”

(Stuewe and Sons Inc., Tangent, OR) containing greenhouse soil (Potting Mix, Lawn

Products Inc., Marysville, OH). Six days after planting, 10 uniform seedlings of each accession were selected and randomized in an alpha-lattice design (described below) distributed among four 30.5 x 30.5 cm racks (Stuewe and Sons Inc., Tangent, OR), each holding 90 seedlings. Each rack was placed into a dacron cage (Louie and Anderson,

1993) along with 10 ‘Spirit’ seedlings. Then, 500 viruliferous D. maidis were introduced in each cage for an inoculation access period (IAP) of seven days in a growth chamber

(12 h light/dark periods at 25°C, with 600 µmol m-2 sec-1). After the IAP, plants were fumigated and transferred to a growth chamber under similar conditions for symptom development.

2.3.4. Symptom evaluation

Following the end of the IAP, which was defined as 0 days post inoculation (dpi), disease incidence and severity were observed daily and recorded at 7, 14 and 21 dpi.

Incidence was estimated based on the number of plants for each accession showing symptoms, and severity was evaluated on the two uppermost leaves of individual plants using a six-point scale: 0, no symptoms; 1, chlorotic spots covering < 25% of the leaf

47 surface; 2, bright chlorotic spots and short stripes covering 25 - 50% of the leaf surface;

3, bright chlorotic spots and stripes on 50 - 75% of the leaf surface; 4, bright chlorotic spots and stripes covering more than 75% of the leaf surface; and 5, a previously symptomatic plant that died (Fig. 2.1). The area under disease progress curve (AUDPC) was calculated from the incidence data on the 10 plants of each genotype at 7, 14, and 21 days after IAP. Disease severity indices (DSI) were calculated from incidence and severity data at 21 dpi using the formula:

( ) (Grau et al., 1982).

2.3.5. MRFV detection with ELISA

The presence of MRFV in putatively resistant accessions was evaluated using enzyme-linked immunosorbent assay (ELISA). Ten seedlings of each accession were inoculated and evaluated for symptom development as outlined above. B73, Spirit, Oh28 and Saint Croix were included as susceptible and resistant controls. At 21 dpi, leaf samples (0.15 g) were taken from the youngest expanding leaf, pooled by accession and stored at –20 °C prior to analysis. PAS-ELISA was carried out as previously described

(Edwards and Cooper, 1985), using MRFV antisera (Bradfute et al., 1979) at a 1:2000 dilution. Absorbance of the samples and the concentration of virus were determined as previously described for Maize fine streak virus (Todd et al., 2010). The experiment was replicated four times.

48

2.3.6. Experimental design and data analysis

An incomplete block design (alpha-lattice) (Barreto et al., 1997; Patterson and

Williams, 1976) with three replications was used to evaluate the incidence, severity, and

AUDPC of MRFV for the 36 maize accessions. To accommodate the size of transmission cages, each replication was divided into four incomplete blocks containing 10 individuals for each of nine accessions plus 10 susceptible controls. Analysis of variance (ANOVA) for incidence, severity, AUDPC, and DSI were calculated using the PROC MIXED function of SAS (SAS Institute, Inc., Cary, NC). Maize accession was a fixed factor, and replicate and block were treated as random factors. ELISA responses were analyzed using PROC GLM. Means separations between genotypes were estimated using Tukey’s test (P=0.05). Pearson correlation analysis was used to assess the relationship between the variables in pair-wise comparisons.

2.3.7. Inheritance of resistance

To evaluate the inheritance of resistance to MRFV, 40 F1 and 100 F2 seedlings from single ears derived from a Oh28 x Oh1VI cross, as well 30 Oh28 and Oh1VI seedlings were inoculated and evaluated for symptom incidence and severity as outlined above. Data presented are for two independent replications, with seedlings that died without developing disease symptoms being removed from the analysis. For disease incidence in the F2 population, a Chi square test was conducted to assess significance of

Mendelian segregation ratios.

49

2.4. Results

2.4.1. Responses of maize accessions to inoculation with MRFV

The first disease symptoms in susceptible control genotypes were observed between four and five dpi. A large proportion (75% and 77%, respectively) of Spirit and

B73 seedlings became symptomatic, with a disease severity rating of two at seven dpi

(Fig. 2.1 and 2.2). Incidence and severity were similar in both susceptible lines at all rating dates, indicating the disease transmission protocol was effective. Disease incidence and severity in susceptible controls did not increase after 7 dpi 14 dpi, respectively (Fig.

2.2). For all lines, incidence and severity were rated up to 21 dpi, in order to detect late developing infections. However, problems with longer-term maintenance of the plants under the growth chamber conditions required by APHIS permits prevented ratings beyond 21 dpi. Incidence and severity at 7, 14, and 21 dpi were significantly correlated (p

< 0.0001), suggesting this period was sufficient for disease evaluation.

There was significant genetic variation (p < 0.001) among the 36 maize accessions at 21 dpi for incidence, severity, AUDPC and DSI (Table 2.1). The residual plots of the variance indicated a linear relationship between the observed and predicted residuals, and no clear pattern for the plots involving standardized residuals. In addition, distribution of variable means for the accessions resembled a normal distribution (data not shown). Thus, homogeneity of variance and normal distribution were assumed.

Although adjusted means were estimated from the alpha lattice design, the means for three replications are presented since there was no difference in the ranking of the resistant genotypes using calculated or adjusted means. Incidence, severity, AUDPC, and

50

DSI were highly correlated (p < 0.001), with correlation coefficients ranging from 0.6 to

0.9, suggesting that the most susceptible and most resistant accessions would be identified using incidence, severity, AUDPC or DSI (data not shown). In particular,

AUDPC and DSI were highly correlated (correlation coefficient of 0.95, p < 0.0001). The ranking of resistant accessions for these two traits did not differ (Table 2.1), so further discussion will be limited to the AUDPC trait.

Most of the accessions became infected with MRFV. While the susceptible controls B73 and Spirit had high symptom incidence and severity, fewer than 5% of seedlings in accessions previously identified as resistant (INIAP-180, CML459, and Saint

Croix) became symptomatic, and severity ratings for these lines were ≤1.0. Similarly, susceptible controls had AUDPC values of 36.0 and resistant controls had AUDPC values of 1.8 or less. Thus, the controls behaved as predicted from previous disease resistance tests (Bustamante et al., 1998; Toler et al., 1985; Vandeplas, 2003). Disease incidence in CML103, Ms71, Oh28, CML228, Pool12, HP301, P39, CML247, Tx303,

M37W, Ki3, Oh7B, NC350, IL14H, NC358, CML277, B97, M162W, CML322, and

Pool11 was similar to that in susceptible controls (p > 0.05). Disease incidence, severity and AUDPC scores ranged from 30 to 96%, 2.1 to 4.3, and 10.6 to 36.4, respectively, in this group of accessions. Although CML202, Tzi8, DR, Mo18W, CML69, CML52, and

CML333 had significantly lower disease incidence and AUDPC scores than the susceptible controls, disease severity in these accessions was similar to the susceptible controls (Table 2.1). In contrast, in accessions Ki11, INIAP-180 and Oh1VI symptom incidence was less than 10%, mean severity ratings were less than 0.7 and AUDPC scores

51 were less than 2.1, significantly lower than those in the susceptible controls. Further,

CML287, CML459, Cuba, and Saint Croix developed no disease symptoms during the

21-day rating period.

A semi-quantitative PAS-ELISA was used to confirm that MRFV was not present in putatively resistant lines. Virus concentrations in the upper non-inoculated leaves indicated significant MRFV concentrations in the susceptible controls (Fig. 2.3). In contrast, CML287, Cuba, Oh1VI, and Saint Croix were similar to non-inoculated control plants in this assay. These results indicated that virus was not detected in accessions with few or no symptoms.

2.4.2. Inheritance of resistance to MRFV in Oh1VI

Only a few seedlings of F1 plants derived from a cross of Oh1VI x Oh28 developed limited disease symptoms during the 21 day evaluation period after MRFV inoculation (Table 2.2). In these experiments, 23% of F2 seedlings developed symptoms.

The mean severity ratings for symptomatic F1 and F2 plants were 1.7 and 2.8, respectively. Infection and disease severity in the parental lines were similar to the previous experiment (Table 2.1) with Oh1VI developing no disease symptoms, and 61% of Oh28 seedlings developing symptoms with a severity rating of 3.3 in symptomatic plants (Table 2.2). For 200 F2 seedlings inoculated over two replications, 18 died prior to

21 dpi without developing disease symptoms, and these were removed from the analysis.

Of the remaining plants, 140 developed no symptoms and 42 developed symptoms with an average severity rating of 2.8. The observed healthy: symptomatic ratio was consistent

52 with a 3:1 or 13:3 segregation ratio in Chi-square tests (p-value >0.1), but differed from

15:1, 7:5, and 9:7 segregation ratios (p-value <0.0001).

2.5. Discussion

MRFV is one of the most important virus diseases affecting maize in the

Americas, from Argentina to the southern United States (Gordon and Thottappilly, 2003;

Kogel et al., 1996; Redinbaugh and Pratt, 2009). The impact of the disease is greater in the highlands of Mexico, Central and South America where it is usually present as a disease complex called “achaparramiento” or red stunt (Vandeplas, 2003). The complex is caused by the presence MRFV and two additional pathogens, CSS and MBSP (Gordon and Thottappilly, 2003; Hammond and Bedendo, 2001). A number of CSS- and MBSP- resistant cultivars are available (Carpane, 2007; Silveira et al., 2008; Vandeplas, 2003), but genotypes resistant to MRFV are scarce. Only a few MRFV-resistant lines have been identified, and the genetic basis of resistance has not been described (Bustamante et al.,

1998; Vandeplas, 2003). One reason for this lack of information is the heterogeneous nature of previously identified sources of resistance. For example, Saint Croix (landrace) and INIAP176 (open pollinated) are outcrossing populations, making genetic analyses difficult, and no recombinant populations derived from inbred lines (e.i., CML459) suitable for genetic analysis are available. In this study, we identified several new sources of MRFV-resistance in maize germplasm, and determined that resistance in one inbred line is likely to be the result of one or a few resistance genes.

53

The susceptible responses of B73 and Spirit to inoculation with MRFV were expected based on previous results indicating little resistance to the virus in temperate germplasm (Louie et al., 2000; Toler et al., 1985). MRFV resistance in Saint Croix and

CML459 was confirmed, with no symptoms developing on either genotype under growth chamber conditions. Comparison of these results with those of previous studies, in which relatively high ELISA values were reported in Saint Croix and a few plants developed mild symptoms CML459 late in infection (Vandeplas, 2003), suggest that environment plays a role in MRFV resistance in these accessions.

Most of the lines tested were susceptible to MRFV, with incidence ranging from

20 to 96%. The tropical lines Ki11 and CML333, that were included as founders of the

NAM population, had an intermediate response to MRFV inoculation with disease incidence of 10 and 14%, respectively (Table 2.1); however, disease severity in CML333 was not different from the susceptible controls. It remains to be determined whether these intermediate responses are a result of partial resistance to the virus, resistance to insect inoculation, or some mechanism that reduced transmission rates in these lines. Although

Ki11 x B73 and CML333 x B73 populations could be used to map the location of loci conferring resistance, the intermediate responses to MRFV inoculation could reduce the power for QTL detection. Additionally, incomplete resistance phenotypes are usually conditioned by many loci with small effects (Poland et al., 2009), which are less amenable to manipulation in a breeding context. Nonetheless, the availability of a linkage maps, genotypic data, and available recombinant inbred populations derived from these

54 lines could be valuable for understanding MRFV resistance, especially in combination with mapping in other populations derived from highly resistant lines.

Novel sources of resistance to MRFV were identified in the inbred lines Oh1VI,

CML287 and Cuba were highly resistant to MRFV with ELISA responses and symptom development similar to healthy non-inoculated control plants (Fig. 3). Interestingly, all lines identified as highly resistant in this study were derived from germplasm originating in the Caribbean. Oh1VI and Saint Croix were developed or collected from the Virgin

Islands (Bustamante et al., 1998; Louie et al., 2002), and Cuba was derived from a Cuban open pollinated population (Redinbaugh, unpublished results). CML287 is derived from

CIMMYT population 24, originally developed from germplasm collected in eastern

Mexico and Antigua (CIMMYT, 1981). INIAP-180 was derived from the MRFV- resistant open pollinated variety INIAP-176 (Bustamante et al., 1998), and has germplasm from the highlands of El Salvador, Guatemala and Honduras, in its genetic background (Caviedes, 1986). Notably, Oh1VI is resistant to Maize chlorotic dwarf virus

(Jones et al., 2004), Maize dwarf mosaic virus and Sugar cane mosaic virus (Jones et al.,

2007), Maize fine streak virus (Redinbaugh et al., 2002) and Maize necrotic streak virus

(Louie et al., 2000). These observations suggest the importance of Central American and

Caribbean maize germplasm as sources of resistance to viral diseases.

Resistance to MRFV conferred by Oh1V1 was successfully transmitted to F1 and

F2 progenies. Symptom development in F1 plants was similar to the resistant parent, suggesting resistance may be dominant. Resistance segregated in the F2 population consistently with 3:1 and 13:3 segregation ratios (Table 2.2). However, because of the

55 relatively small number of F2 plants tested, it is possible that disease escapes may have interfered with the determining the ratio of resistant to susceptible plants. If MRFV transmission to F2 plants was the same as that observed for Oh28 (61%), and we therefore assumed a disease escape rate of 39%, then the observed segregation would fit both 7:5 and 9:7 segregation ratios (p > 0.1). While it is not possible to identify the number of genes involved in MRFV in resistance from these experiments, our results suggest that one or two genes models are sufficient to explain the resistance conferred by Oh1V1.

Further analysis to identify and map gene(s) conferring resistance to MRFV will be required to provide more definitive information.

It is not known whether the resistance sources identified in this study will be effective against all described MRFV isolates (Chicas et al., 2007). However, previously identified MRFV-resistant lines had similar responses to the U.S. isolate used in this study, and the Mexican or Costa Rican MRFV isolates used in other studies (Bustamante et al., 1998; Vandeplas, 2003). The isolate used for these studies was originally collected from Texas, where MRFV isolates were subsequently shown to belong to the same phylogenetic group as those from Mexico and Central America (Chicas et al., 2007), and this could explain the similar responses. It would be of interest to determine whether resistance to phylogenetically distinct isolates from South America follows a similar pattern. In summary, we have confirmed previously reported sources of resistance to

MRFV and identified three resistant maize inbred lines that could be directly used in national programs for development of maize hybrids or open pollinated varieties with increased resistance to MRFV.

56

2.6. Acknowledgments

I would like to thank M. Redinbaugh and D. Francis for contributing to this chapter which was accepted for publication in Plant Disease. I thank M. Jones (USDA-

ARS) for valuable discussion and for providing seeds of inbred lines and F1 and F2 populations. I thank S. Taba from CIMMYT for helpful discussion and for providing seed. I also thank C. Gardner from NCRPIS for seed of the NAM founder lines, and

Michele Gardiner (Syngenta Seeds Inc.) for Spirit sweet corn seed. I am grateful to

Charlie Summers, UC-Riverside, CA for providing the Dalbulus maidis, to J. Todd

(USDA-ARS) for maintaining the insect colony, and K. Willie (USDA-ARS) for technical assistance. I thank the Instituto Nacional Autónomo de Investigaciones

Agropecuarias (INIAP), Ecuador for a fellowship to support my Ph.D. study.

57

2.7. References

Barreto, H., Edmeades, G., Chapman, S., and Crossa, J. 1997. The alpha lattice design in plant breeding and agronomy: generation and analysis. Pages 544-551 in: Developing Drought and Low N-tolerant Maize. in: Edmeades, G., Banziger, M., Mickelson, H., andPeña-Valdivia, C., eds.CIMMYT, Mexico DF.

Bradfute, O.E., Toler, R.W., Boothroyd, C.W., Robertson, D.C., Nault, L.R., and Gordon, D.T. 1979. Identification of Maize rayado fino virus in the United States. Plant Dis 64:50-53.

Bustamante, P.I., Ramirez, P., and Hammond, R. 1998. Evaluation of maize germ plasm for resistance to Maize rayado fino virus. Plant Dis 82 1:50-56.

Carpane, P.D.,. 2007. Host resistance and diversity of Spiroplasma kunkelii as components of corn stunt disease.

Caviedes, M. 1986. INIAP-180: Nueva variedad de maíz de alto rendimiento. INIAP, Boletin divulgativo 180.

Chicas, M., Caviedes, M., Hammond, R., Madriz, K., Albertazzi, F., Villalobos, H., and Ramírez, P. 2007. Partial characterization of Maize rayado fino virus isolates from Ecuador: Phylogenetic analysis supports a Central American origin of the virus. Virus Res 126 1-2:268-276.

CIMMYT. 1981. Report on maize improvement: 1980-81.

De-Oliveira, E., Duarte, A.P., De-Carvalho, R., and De-Oliveira, A.C. 2004. Molicutes e virus na cultura do milho no Brasil: Caracterizacao e factores que afetam sua incidencia. Pages 17 in: Doencas en Milho. Molicutes, Virus, Vetores e Mancha por Phaeosphaeria. in: De-Oliveira, E., andDe-Oliveira, C.M., eds.Embrapa Informacao Tecnologica, Brasilia.

Edwards, M.L., and Cooper, J.I. 1985. Plant virus detection using a new form of indirect ELISA. J Virol Methods 11 4:309-319.

Espinoza, A.M., and Gamez, R. 1980. La ultraestructura de la superficie foliar de cultivares de maíz infectados con el virus del rayado fino. Turrialba 30 4:413-420.

Gamez, R. 1983. The ecology of Maize rayado fino virus in the American tropics. Pages 268-274 in: Plant Virus Epidemiology. in: Plumb, R.T., andThresh, J.M., eds.Blackwell Scientific Publications, Oxford.

58

Gamez, R. 1969. A new leafhopper-borne virus of corn in Central America. Plant disease reporter 12:929-932.

Gamez, R., and Leon, P. 1988. Maize rayado fino and related viruses. Pages 213-233 in: Polyhedral virions with monopartite RNA genomes / edited by Renate Koenig. New York : Plenum Press, c1988.

Gamez, R., and Leon, P. 1985. Ecology and evolution of a Neotropical leafhopper-virus - maize association. Pages 331-349 in: The leafhoppers and planthoppers. in: Nault, L.R., and Rodriguez, J.G., eds.John Wiley & Sons Inc., New York.

Gordon, D.T., and Thottappilly, G. 2003. Maize and Sorghum. Pages 295-334 in: Virus and virus-like diseases of major crops in developing countries. in: Loebenstein, G., andThottappilly, G., eds.Kluwer Academic Publishers, The Netherlands.

Grau, C.R., Radke, V.L., and Gillespie, F.L. 1982. Resistance of soybean cultivars to Sclerotinia sclerotiorum. Plant Dis 66:506-508.

Hammond, R.W., and Bedendo, I.P. 2001. Role of Maize rayado fino virus in the etiology of "red stunt" disease in Brazil. Plant Dis 85 1:99-99.

Hammond, R., and Ramirez, P. 2001. Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus. Virology 282 2:338-347.

Jones, M.W., Redinbaugh, M.G., Anderson, R.J., and Louie, R. 2004. Identification of quantitative trait loci controlling resistance to Maize chlorotic dwarf virus. Theor Appl Genet 110:48-57.

Jones, M.W., Redinbaugh, M.G., and Louie, R. 2007. The Mdm1 locus and maize resistance to Maize dwarf mosaic virus. Plant Dis 91:185-190.

Kogel, R., Ramirez, P., and Hammond, R.W. 1996. Incidence and geographic distribution of Maize rayado fino virus (MRFV) in Latin America. Plant Dis 80 6:679-683.

Louie, R., and Anderson, R.J. 1993. Evaluation of Maize chlorotic dwarf virus resistance in maize with multiple inoculations by Graminella nigrifrons (Homoptera: Cicadellidae). J Econ Entomol 86:1579-1583.

Louie, R. 1995. Vascular puncture of maize kernels for the mechanical transmission of maize white line mosaic-virus and other viruses of maize. Phytopathology 85:139-143.

Louie, R., Abt, J.J., Anderson, R.J., Redinbaugh, M.G., and Gordon, D.T. 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family Tombusviridae. Plant Dis 84:1133-1139. 59

Louie, R., Redinbaugh, M.G., Anderson, R.J., and Jones, M.W. 2002. Registration of maize germplasm Oh1VI. Crop Sci 42:991-991.

Madriz-Ordenana, K., Thordal-Christensen, H., Collinge, D.B., Ramirez, P., Rojas- Montero, R., and Lundsgaard, T. 2000. Mechanical transmission of maize rayado fino marafivirus (MRFV) to maize and barley by means of the vascular puncture technique. Plant Pathol 49:302-307.

McMullen, M.D., Kresovich, S., Villeda, H.S., Bradbury, P., Li, H., Sun, Q., Flint- Garcia, S., Thornsberry, J., Acharya, C., Bottoms, C., Brown, P., Browne, C., Eller, M., Guill, K., Harjes, C., Kroon, D., Lepak, N., Mitchell, S.E., Peterson, B., Pressoir, G., Romero, S., Rosas, M.O., Salvo, S., Yates, H., Hanson, M., Jones, E., Smith, S., Glaubitz, J.C., Goodman, M., Ware, D., Holland, J.B., and Buckler, E.S. 2009. Genetic properties of the maize nested association mapping population. Science 325:737-740.

Nault, L.R., and Bradfute, O.E. 1979. Corn stunt: involvement of a complex of leafhopper-borne pathogens. Pages 561-586 in: Leafhopper vectors and plant disease agents. in: Maramorosch, K., andHarris, K.F., eds.Academic Press, New York.

Nault, L.R., Gordon, D.T., and Gingery, R.E. 1980. Leafhopper transmission and host range of Maize rayado fino virus. Phytopathology 70:709-712.

Patterson, H.D., and Williams, E.R. 1976. A new class of resolvable incomplete block designs. Biometrika 63:83-92.

Poland, J.A., Pratt, R.C., Nelson, R.J., Balint-Kurti, P., and Wisser, R.J. 2009. Shades of gray: the world of quantitative disease resistance. Trends Plant Sci 14:21-29.

Ramirez-Rojas, S., Romero-Rosales, F., Jeffers, D., Martinez Garza, A., and Mejia Andrade, H. 1988. Reacción de ocho variedades de maíz al virus del rayado fino en Chapingo, México. Agricultura Tecnica en Mexico 24:11-18.

Redinbaugh, M.G., Houghton, W., Salomon, R., Creamer, R., Hogenhout, S.A., Gordon, D.T., Ackerman, J., Meulia, T., Seifers, D.L., Abt, J.J., Styer, W.E., and Anderson, R.J. 2002. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathology 92:1167-1174.

Redinbaugh, M.G., and Pratt, R.C. 2009. Virus Resistance. Pages 251-268 in: Handbook of maize: Its Biology. In: Bennetzen, J.L., andHake, S.C., eds.Springer, New York.

Silveira, F.T., Moro, J.R., da Silva, H.P., de Oliveira, J.A., and Perecin, D.O.L. 2008. Inheritance of the resistance to corn stunt. Pesquisa agropecuária brasileira. 43:1717- 1724.

60

Todd, J.C., Hoy, C., Hogenhout, S.A., Ammar, E., and Redinbaugh, M.G. 2010. Plant Host Range and Leafhopper Transmission of Maize fine streak virus. Phytopathology 100:1138-1145.

Toler, R.W., Harris, K.F., Bockholt, A.J., and Skinner, G. 1985. Reactions of maize (Zea mays) accessions to Maize rayado fino virus. Plant Dis 69:56-57.

Valdez, M., Madriz, K., and Ramírez, P. 2004. A method for genetic transformation of maize for resistance to viral diseases. Rev Biol Trop 52:787-793.

Vandeplas A. 2003. Evaluation of sixty highland elite maize genotypes for resistance to Maize rayado fino virus. The Katholieke Universiteit Leuven. Belgium.

Vasquez, J., and Mora, E. 2007. Incidence of and yield loss caused by Maize rayado fino virus in maize cultivars in Ecuador. Euphytica 153:339-342.

Yu, J., Buckler, E.S., McMullen, M.D., and Holland, J.B. 2008. Genetic design and statistical power of nested association mapping in maize. Genetics 178:539-511.

61

a b Disease symptoms score Accession Source e Incidence (%) Severityc AUDPCd DSI CML103 N 96 3.0 36.4 57.9 B73 N 80 2.7 36.0 41.3 Spirit S 78 3.0 36.0 48.1 Ms71 N 70 3.3 34.1 46.7 Oh28 U 69 3.0 23.4 40.5 CML228 N 67 2.0 18.8 26.0 Pool12 C 63 2.2 22.2 28.0 HP301 N 62 3.8 27.6 48.8 P39 N 59 3.0 18.2 39.2 CML247 N 54 3.2 12.9 33.1 Tx303 N 53 3.3 17.7 37.6 M37W N 52 2.7 24.0 40.3 Ki3 N 50 2.2 12.1 23.0 Oh7B N 48 3.2 13.2 31.7 NC350 N 47 3.0 13.2 28.0 Il14H N 44 4.3 20.6 38.9 NC358 N 39 2.9 15.1 23.6 CML277 N 35 2.9 10.5 25.1 B97 N 32 3.6 10.2 22.7 M162W N 30 4.0 12.6 22.4 CML322 N 30 2.1 10.6 12.7 Pool11 C 30 3.0 12.7 18.0 CML202 C 27 1.1 5.4 6.1 Tzi8 N 25 2.0 4.8 10.2 DR U 24 1.0 3.2 4.8 Mo18W N 23 3.3 8.5 16.7 CML69 N 22 1.1 3.7 7.0 CML52 N 21 2.5 5.4 9.3 CML333 N 14 2.2 3.2 6.0 Ki11 N 10 0.4 2.1 2.8 INIAP-180 C 4 0.7 1.8 1.8 Oh1VI U 3 0.3 0.6 0.7 CML287 C 0 0.0 0.0 0.0 Continued…

Table 2.1. Responses of maize accessions to Maize rayado fino virus (MRFV) inoculation.

62

Table 2.1 Continued

CML459 C 0 0.0 0.0 0.0 Cuba U 0 0.0 0.0 0.0 Saint Croix N 0 0.0 0.0 0.0 Mean 38 2.2 13.2 22.1 Tukeyf 51 2.9 20.9 40.8 aDisease symptoms scored at 21 days post inoculation (dpi). Data presented are the means of three replications with 10 plants per replication. bSources of seeds. N, North Central Regional Plant Introduction Station (Iowa); C, International Maize and Wheat Improvement Center (Mexico); S, Syngenta (Idaho); U, USDA-ARS Corn, Soybean and Wheat Quality Research Unit (Ohio). cSeverity was evaluated on a scale 0 to 5, where 0 = no disease symptoms and 5 = dead after being shown disease symptoms (Fig. 1). dAUDPC, area under the disease progress curve calculated from the incidence values scored at 7, 14 and 21 dpi, as outlined in the materials and methods. eDSI, disease severity index at 21 dpi. fMinimum significant difference according to Tukey’s studentized range test (P = 0.05).

Table 2.1. Responses of maize accessions to Maize rayado fino virus (MRFV) inoculation.

63

Genotype Na Incidence Severity Severity classd b c (%) (0 – 5) 0 1 2 3 4 5 F1 3 / 80 4 (0 – 8) 1.7 77 1 2 F2 42 / 182e 23 (15 – 31) 2.8 140 11 29 2 Oh1VI 0 / 50 0 (0 – 0) 0.0 50 Oh28 35 / 58 61 (60 – 61) 3.3 23 1 25 7 2 aNumber of seedlings with disease symptoms / total number of seedlings evaluated in two independent replications. bThe proportion of plants showing any disease symptom at 21 dpi. The data presented are the mean (range) from two replications. cSymptom severity at 21 dpi on a 0 to 5 scale, where 0 = no disease symptoms and 5 = dead after developing disease symptoms (Fig. 1). The data presented are the mean of symptomatic plants. dNumber of individuals in each severity class at 21 dpi. eFit 3:1 and 13: 3 resistant: susceptible ratios according to Chi square test (p>0.05).

Table 2.2. Inheritance of resistance to Maize rayado fino virus (MRFV) in F1 and F2 maize populations derived from the resistant Oh1V1 and the susceptible Oh28.

64

Fig. 2.1. Symptom severity scale used to evaluate the response of maize seedlings to inoculation with Maize rayado fino virus.

65

Fig. 2.2. Maize rayado fino virus disease progress in susceptible maize accessions. Symptoms were evaluated in maize ‘Spirit’ and B73 at 7, 14, and 21 days post inoculation (dpi). Data presented are the means ± s.d. for three replications with 10 plants per replication. A, Incidence and. B, Mean severity rating of symptomatic plants.

66

Fig. 2.3. Maize rayado fino virus antigen in maize leaves estimated with semi- quantitative PAS-Enzyme-linked immunosorbent assay. Titer was estimated in selected maize lines at 21 days post inoculation. Data presented are the mean ± s.d. Bars labeled with same letter are not significantly different (Tukey test, P = 0.05).

67

CHAPTER 3

GENETIC ANALYSIS OF RESISTANCE TO SIX VIRUS DISEASES IN A

MULTIPLE VIRUS-RESISTANT MAIZE INBRED LINE

3.1. Abstract

Virus diseases in maize can cause severe yield reductions that threaten crop production and food supplies in some regions of the world. Genetic resistance to different viruses has been characterized in maize populations under diverse environments and screening techniques, and resistance loci have been mapped to all maize chromosomes.

The maize inbred line, Oh1VI, is resistant to at least 10 viruses, including viruses in five different families. To determine the genes and inheritance mechanisms responsible for the multiple virus resistance in this line, F1 hybrids, F2 progeny and a recombinant inbred line (RIL) population derived from a cross of Oh1VI and the virus-susceptible inbred line

Oh28 were evaluated. Progeny were screened for their responses to Maize dwarf mosaic virus, Sugarcane mosaic virus, Wheat streak mosaic virus, Maize chlorotic dwarf virus,

Maize fine streak virus, and Maize mosaic virus. Depending on the virus, dominant, recessive, or additive gene effects were responsible for the resistance observed in the F1.

For all viruses, one, two or three gene models explained the observed segregation of 68 resistance in the F2 generation. Composite interval mapping in the RIL population identified 17 resistance QTLs associated with the six viruses. Of these, 15 were clustered in specific regions of chromosomes 2, 3, 6, and 10. It is unknown whether these regions of clustered QTLs contain single or multiple virus resistance genes, but the coupling phase linkage of genes conferring resistance to multiple virus diseases in this population could facilitate breeding efforts to develop multi-virus resistant crops.

3.2. Introduction

Maize is a natural host for more than 50 viruses and an experimental host for about 30 more (Lapierre and Signoret, 2004), but only some cause diseases that seriously affect yield (Ali and Yan, 2012; Redinbaugh and Pratt, 2009). Among the most damaging are members of the Potyviridae and Maize chlorotic mottle virus (MCMV), which form the devastating complex known as maize lethal necrosis virus (Uyemoto et al., 1980;

Wangai et al., 2012). Plants have evolved passive and active defense mechanisms that are responsible for the suppression of virus multiplication and spread. Such mechanisms require interaction of plant and viral factors to confer plant resistance or susceptibility

(Fraser, 1990; Gomez et al., 2009; Kang et al., 2005). Identifying the loci conferring resistance to virus diseases offers an approach to develop genetically resistant lines that are able to reduce the yield losses caused by existing and emerging viral diseases.

Viruses in at least eight different families have caused significant agronomic losses in maize (Ali and Yan, 2012; Redinbaugh and Pratt, 2009). Distributed worldwide, potyviruses are the most common and most studied viruses of maize (Ali and Yan, 2012;

69

Gordon et al., 1981; Gordon and Thottappilly, 2003; Lapierre and Signoret, 2004).

Potyviruses are single stranded positive sense monopartite RNA viruses with a single open reading frame encoding a polyprotein that is post-translationally processed into at least 10 mature proteins (Lapierre and Signoret, 2004; Stenger et al., 1998). Maize- infecting members of the family Potyviridae include Maize dwarf mosaic virus (MDMV),

Sugar cane mosaic virus (SCMV) and Wheat streak mosaic virus (WSMV) (Ali and Yan,

2012; Jones et al., 2011; Shulka et al., 1994; Sun et al., 2010; Williams and Pataky,

2012). In nature, MDMV and SCMV are nonpersistently transmitted by aphids (Nault and Knoke, 1981) and WSMV is semi-persistently transmitted by mites (Slykhuis, 1955).

Rhabdoviruses are single stranded RNA negative sense (non-coding) viruses with a monopartite genome and five major structural proteins. The rhabdovirus Maize mosaic virus (MMV) causes an important disease of maize in regions of Africa, South America,

Hawaii and Australia (Gordon et al., 1981; Ming et al., 1997; Redinbaugh and Pratt,

2009; Thottappilly et al., 1993). Maize fine streak virus (MFSV) is a phylogenetically distinct rhabdovirus that was recently found in a maize hybrid field near Bainbridge, GA

(Redinbaugh et al., 2002). MMV and MFSV are persistently and propagatively transmitted by the planthopper (Perigrinus maidis) and the leafhopper (Graminella nigrifrons), respectively (Nault and Knoke, 1981, Redinbaugh et al., 2002).

The waikavirus Maize chlorotic dwarf virus (MCDV) is present the southern and southeastern regions of the United States where it has caused significant problems

(Gordon et al., 1981; Lapierre and Signoret, 2004). MCDV virions contain a monopartite single-stranded positive sense RNA genome that encodes a large post-translationally

70 processed polyprotein (Hull, 2002). MCDV is semi-persistently transmitted by the leafhopper G.nigrifrons (Nault and Knoke, 1981).

Loci conferring resistance to various virus diseases have been reported on all maize chromosomes (Bonamico et al., 2012; McMullen and Simcox, 1995; Melchinger et al., 1998; Redinbaugh et al., 2004; Wisser et al., 2006; Xia et al., 1999). These studies involved different viruses, maize populations, environments and screening techniques.

Resistance loci to the potyviruses MDMV, SCMV, Sorghum mosaic virus (SrMV) and

Johnsongrass mosaic virus (JGMV) and to WSMV cluster in specific regions of chromosomes 3, 6 and 10 (Dussle et al., 2000; Jones et al., 2004; Jones et al., 2007;

McMullen et al., 1994; Prazeres De Souza et al., 2008; Redinbaugh and Pratt, 2009,

Stewart et al., 2012; Zhang et al., 2003). It is unknown whether these QTLs also provide resistance to other unrelated virus diseases whose resistance loci have been less well characterized or never mapped.

Our goal was to determine the inheritance and location of genes conferring resistance to a diverse set of viruses in the multiply virus resistant maize inbred line

Oh1VI (Louie et al., 2002), and to further test whether resistance loci to diverse taxonomic virus families cluster in the aforementioned maize chromosome regions. To accomplish this, the F1 hybrids, F2 progeny and a recombinant inbred line (RIL) population derived from a cross between Oh1VI and the virus-susceptible Oh28 were tested for their independent responses to inoculation with a variety of viruses from three families, including MFSV, MMV, MCDV, MDMV, SCMV, and WSMV. The RIL plants

71 were genotyped, a genetic map was built, and the positions of resistance loci were determined.

3.3. Materials and methods

3.3.1. Plant material

The inbred line Oh1VI (PI 614734) is a type derived from a Virgin

Island population developed by the USDA-ARS and the OARDC (Louie et al., 2002).

Oh1VI is highly resistant to phylogenetically distinct viruses, including MFSV, MMV,

MCDV, MDMV, SCMV, WSMV, Maize rayado fino virus (MRFV), and Maize necrotic streak virus (MNeSV) (Jones et al., 2004; Louie et al., 2000; Redinbaugh et al., 2002;

Zambrano et al., 2013). Oh28 {(CI.112-1 x Oh920) x (I11.A x I11.B)} is a yellow dent corn released in 1943 (Pratt, R., personal communication) that is susceptible to all of the viruses mentioned above (Louie et al., 2000; McMullen and Louie, 1989; McMullen et al., 1994; Redinbaugh et al., 2002). F1 hybrids, F2 progeny and RIL families were generated from the cross between Oh1VI and Oh28. The F1 was made in the summers of

1996 and 2003. Through 2006, 511 F2 ears were generated. Seeds of F2 plants were planted ear to row, and successively self-pollinated every year. By 2010, 260 RILs were self pollinated between seven to nine times without selection. The lines have been maintained by the Corn, Soybean and Wheat Quality Research Unit (CSWQRU) at the

OARDC.

72

3.3.2. Viruses and vectors.

The MDMV, SCMV, WSMV, and MCDV-severe isolates were collected in Ohio

(Hunt et al., 1988; Louie, 1986). The MFSV and MMV isolates were collected in Georgia and Hawaii, respectively (Ming et al., 1997; Redinbaugh et al., 2002). Virus identity was verified by enzyme-linked immunosorbent assay (ELISA) and bioassay (for MDMV,

SCMV and WSMV) as previously described (Jones et al., 2007). The MCDV, MMV and

MFSV isolates were maintained by serial transmission using vascular puncture inoculation (Louie, 1995; Redinbaugh et al., 2001) or insects (Louie and Anderson, 1993;

Todd et al., 2010), while MDMV, SCMV, and WSMV were maintained by serial transmission using rub-inoculation on susceptible maize (Oh28) by the CSWQRU at the

OARDC.

Graminella nigrifrons, the vector of MFSV and MCDV, was collected from fields near Wooster, OH. Peregrinus maidis, the vector of MMV, was a gift of Dr. William

Belote (Dupont, Stine-Haskell Research Center, Newark, DE). Colonies of G. nigrifrons and P. maidis were maintained on oat ‘Armor’ and sweet corn ‘Early Sunglow’

(Schlessman Seed Co. Milan, OH) seedlings, respectively, in growth chambers with a

25oC – 15 h light period (800 µmol m-2 sec-1) and a 25oC – 9 h dark period.

Viruliferous MFSV-G. nigrifrons and MMV-P. maidis were obtained by allowing virus-naive nymphs to feed on virus-infected plants for 26 days before being used for inoculation (Todd et al., 2010). Inoculation access and latency periods of 7 and 19 days, respectively, were used as previously described (Zambrano et al., 2013). Viruliferous

73

MCDV-G.nigrifrons were obtained by feeding virus-naive adults on infected MCDV plants for two days before being used for inoculation (Louie and Anderson, 1993).

3.3.3. Inheritance of the resistance

Seedlings of the F1 and F2 generations of the cross between Oh1VI and Oh28 were independently inoculated with the six viruses to determine the inherence of the resistance. Experiments using MFSV, MMV, and MCDV were conducted in growth chambers and greenhouses, and those for MDMV and SCMV were conducted at the

OARDC Snyder Farm (Wooster, OH) during the summers of 2006 and 2010. The screening with WSMV was conducted in the same field location during the summer of

2010. The environments for these experiments (growth chambers, greenhouses, or field) were determined, in part, by the conditions of USDA, Animal and Plant Health

Inspection Service permits for working with the viruses.

For the evaluation of MFSV, MCDV, and MMV, 100 F2 and 60 F1 six-day old seedlings were randomized and divided between two Dacron covered cages each containing 500 viruliferous vector insects. Each cage also contained ten seedlings of the resistant and the susceptible parents as controls. The cages were then moved to a growth chamber (12 h light/dark periods with 600 µmol m-2 sec-1 at 25°C) for a seven-day inoculation access period (IAP). For MCDV, the multiple inoculation method described by Louie and Anderson (1993) was used. After the IAP, plants were fumigated and transferred to a greenhouse for symptom development. The greenhouse temperature was set at 25oC and 18oC (day and night, respectively) and natural light was supplemented

74 with 400W high-pressure sodium lights (P. L. Light System, Beamsville, ON, Canada) between October and April to obtain a 12 h light period. These experiments were conducted at different times during 2011 and 2012. Disease incidence and severity were recorded at 21 dpi, with the end of the IAP defined as 0 days post inoculation (dpi).

Incidence was determined as the ratio of symptomatic plants/total number of plants, and severity was evaluated on the uppermost expanded leaf of individual plants using a 3- point scale, where 1 = no symptoms, 3 = intermediate symptoms, and 5 = severe disease symptoms (Fig. 3.1).

For field evaluations, plots consisted of 15 to 18 rows of F2 plants bordered by two rows each of Oh1VI and F1 and five rows of Oh28. Each row was planted with 17 seeds in a 3.5 m-long row spaced at 0.76 m. Plots were inoculated with MDMV, SCMV or WSMV four times at two-day intervals beginning at the V2 stage (two leaves present with a visible collar) using a Model H3 Airbrush (Paasche Airbrush Co., Chicago, IL) as previously described (Louie et al., 1983). Disease incidence was scored between 14 and

21 days after the first inoculation as the number of symptomatic plants/total number of plants (Jones et al., 2011).

Phenotypic data for F1 and F2 plants were analyzed relative to parents in order to assess the degree of dominance associated with resistance (Wu et al., 2007). A Chi square test was conducted to assess goodness of fit 3:1, 15:1, 9:7, 11:5, 13:3, and 63:1 segregation ratios for diseases incidence in the F2 generation.

75

3.3.4. RIL phenotypic analysis

The responses of the RILs to inoculation with MFSV, MMV, MCDV and WSMV were evaluated in a greenhouse at the OARDC. For MFSV, MMV, and MCDV, a single seed of each RIL was planted into a “cone-tainer” (16.4 x 2.5 cm; Stuewe and Sons Inc.,

Tangent, OR) containing autoclaved soil. Six days after planting the seedlings were randomly distributed in three racks (30.5 x 30.5 cm; Stuewe and Sons Inc.). Additionally,

10 seedlings of the susceptible parent (Oh28) were placed in each cage as controls.

MFSV, MMV, and MCDV inoculations were conducted using insects as described above. For MMV and MFSV, the experiment consisted of three independent replications, and for MCDV four replications were used. Disease incidence and severity were evaluated as outlined above at 7, 14, and 21 dpi. The area under disease progress curve

(AUDPC) was calculated from the severity data at the three rating dates.

The RIL responses to WSMV were evaluated in a greenhouse as previously described (Jones et al., 2007). Briefly, rows of twelve seeds of each RIL were planted into plastic trays containing autoclaved soil. At 14 days after planting, leaves were rub- inoculated four times at two-day intervals, and symptoms were scored 21 days after the first inoculation. Disease incidence was estimated as percentage of the number of plants with disease symptoms.

The response of the RILs to MDMV inoculation was evaluated in 2009 and 2011, and to SCMV in 2011 and 2012 at Snyder Farm as previously described (Jones et al.,

2011). For each virus, plots consisted of 17 seeds planted in a 3.5 m row spaced at 0.76 for each RIL and were replicated two times in a complete randomized block design.

76

Virus inoculation was conducted using an airbrush as described above. Disease incidence determined from the proportion of plants in each row with disease symptoms at 21 dpi.

Best linear unbiased predictors or BLUPs (Balzarini and Milligan, 2003) for incidence and AUDPC were estimated for each RIL using the PROC MIXED procedure in SAS (version 9.2, SAS Institute Inc., Cary, NC). The model applied was: yij = µ + geni

+ repj + eij; where yij was the phenotypic response value to virus inoculation for the ith genotype in the jth replication, geni represented the individual effect of the ith line, repj was the effect of the jth replication, and eij the residual error term in the model. For

MDMV and SCMV, replication was replaced by year in the model. All factors were considered random. Variance components for disease incidence and AUDPC were estimated by restricted maximum likelihood (REML).

3.3.5. RIL Genotypic analysis

A set of 21 simple sequence repeats (SSR) distributed through the maize genome and known to be polymorphic between Oh1VI and Oh28, and 768 single nucleotide polymorphism (SNP) markers (Jones et al., 2009) were used to genotype the 260 RIL.

Genotyping with SSR markers was conducted as previously described (Jones et al.,

2004), and genotyping with SNP markers was conducted using a marker multiplex assay for the Illumina® BedArray™ platform (Illumina, Inc., San Diego, CA, USA) in the

DuPont Pioneer Marker Laboratory, Johnston, IA.

77

3.3.6. Linkage and mapping analysis

Genetic distances and linkage groups were obtained using JoinMap, ver. 3 (van

Ooijen and Voorrips, 2001) with a minimum LOD threshold of 4. Markers with lower

LOD values were discarded. The segregation of alleles at each locus was tested for the expected 1:1 ratio using a chi-square test (p > 0.05). The Kosambi mapping function was used to convert recombination values to map distances (Kosambi, 1944). Genetic map quality was assessed by comparing the positions of the markers in our map with their positions in the B73 v2 reference genome. The physical position of markers was estimated using BLAST analysis of sequences flanking SNPs (Jones et al., 2009) with the

B73 Reference Genome v2 retrieved from the Maize Sequence webpage

(http://www.maizesequence.org). The position of the SSR markers was obtained from the

Maize Genetics and Genomics Database (http://www.maizegdb.org). Markers whose position could not be estimated by BLAST were placed based on minimizing recombination frequency relative to the nearest BLAST-located marker and compared with its location on the intermated B73 x Mo17 (IBM2) map (Jones et al., 2009).

To identify the region(s) of the genome responsible for the virus resistance as

QTLs in the RIL population, composite interval mapping (CIM) was conducted (Jansen and Stam, 1994) using MapQTL, version 4 (van Ooijen et al., 2002). The significance threshold of the LOD score (p < 0.01) was determined by permutation over each linkage group (Churchill and Doerge, 1994). CIM mapping results for incidence of MFSV, MMV and MCDV were confirmed by single marker regression and a Kruskal-Wallis test

78

(Clewer and Scarisbrick, 2001). Circos software (Krzywinski et al., 2009) was used to display the position of resistance QTLs in the Oh1VI x Oh28 RIL genetic map.

Interactions between each pair of QTLs were determined by two-way ANOVA using the PROC GLM procedure of SAS and Cockerham orthogonal contrast (Kao and

Zeng, 2002) as previously described (Coaker, 2003). To compare the effects on disease incidence of individual markers with their interaction, variance components were estimated by REML.

3.4. Results

3.4.1. Inheritance of the resistance

Virus resistance in Oh1VI was transmitted to the F1 and F2 progeny. As expected,

Oh1VI was completely resistant to MFSV, MMV, MCDV, MDMV, SCMV, and WSMV, with no disease incidence and a severity score of 1 (no disease symptoms). Oh28 was fully susceptible to all the diseases with ≥ 95% infection and disease severity scores of 5

(strong disease symptoms), except for WSMV which had an incidence of 86% (Table

3.1). Few to no seedlings of the F1 generation showed symptoms when inoculated with

MFSV, MMV, MDMV, SCMV, and WSMV. In contrast, 88% of the F1 seedlings displayed disease symptoms when inoculated with MCDV.

Resistance to each of the six viruses segregated in the Oh1VI x Oh28 F2 generation (Table 3.1). The F2 resistant: susceptible segregation ratios were consistent with both 3:1 and 13:3 (p ≥ 0.339) for MFSV, and 9:7 (p = 0.596) for MMV. For MCDV and WSMV, the segregation ratios were consistent with 13:3 (p = 0.083) and 63:1 (p =

79

0.512) ratios, respectively. The segregation for MDMV incidence in F2 plants fit a 15:1 ratio in 2006 (p = 0.501), but not in 2010 (p = 0.041). Segregation of F2 plants for resistance to SCMV was consistent with both 3:1 and 11:5 (p ≥ 0.235) in 2010, but the segregation ratio in 2006 only fit a 13:3 ratio (p = 0.443). These results indicate that one to three major loci could explain the observed segregation patterns for each of the viruses tested.

Most of the susceptible F2 seedlings showed strong disease symptoms for MFSV and MMV, with a mean severity rate ≥ 4 (Table 3.1). From the 20 F2 MFSV-susceptible seedlings and the 39 F2 MMV-susceptible seedlings, 14 and 28, respectively, had a severity score of 5. In contrast, 72 out of the 88 MCDV-susceptible F2 seedlings showed moderate disease symptoms with a mean severity rating of 3.5. These results suggested that resistance to MFSV and MMV had a high dominance component, while MCDV resistance was additive to recessive.

3.4.2. RIL phenotypic analysis

Distribution of disease incidence from the 256 RILs inoculated with MFSV and

MMV resembled a binomial distribution, with a large number of RILs scored either as resistant or susceptible (Fig. 3.2). For example, 105 RIL seedlings inoculated with MFSV did not show disease symptoms in any of the three replications, and 98 RILs showed symptoms in all replications. The remaining 53 lines showed inconsistencies between replications suggesting either the presence of disease escapes or incomplete resistance.

MDMV incidence also resembled a binomial distribution, with 110 RILs with zero

80 incidence and 105 susceptible RILs with 40 to 100% disease incidence. Distribution of

MCDV incidence in the RIL population resembled a right-skewed normal distribution with 132 out of the 256 RILs developing consistent symptoms in all four replications and

17 RILs with no symptoms across all replications. The distribution of SCMV symptoms also resembled a normal distribution with a large number of RILs showing 0% disease incidence. The frequency distribution for WSMV incidence was highly skewed with 212 out of the 256 RILs developing no disease symptoms. Disease incidence in the virus susceptible control line (Oh28) was ≥98% and ≥92% across all the greenhouse and field experiments, respectively.

AUDPC scores for the responses to MFSV, MMV, and MCDV correlated with the disease incidence rates (p < 0.0001), although their AUDPC frequency distributions did not resemble their disease incidence distribution (Fig. 3.3). The percentage of variance explained for MFSV incidence and AUDPC due to genetic effects was 67% and

59%, respectively. The variance due to genetic effects for MMV incidence and AUDPC was 51% and 67%, respectively. For MDMV and SCMV only incidence was measured, and the variance attributed to genetic effects was 81% and 54%, respectively. Disease incidence and AUDPC for MCDV had the lowest percentages of genetic variance with

23% and 30%, respectively. Variance due to replication or year effects for all the traits and virus diseases was ≤ 10% (Table 3.2).

81

3.4.3. Linkage Map

Of the 768 SNP markers evaluated, 275 were polymorphic between Oh1VI and

Oh28. Thirty six polymorphic SNP markers were eliminated from the analysis because of low LOD scores (≤4) for association with their predicted linkage group based on physical location in the B73 reference genome. In addition, four RIL genotypes were excluded from the analysis because the genotyping resulted in >10% missing marker data. With the remaining markers and 256 genotypes, a linkage map spanning a total of 1226 cM was constructed. The map included 10 linkage groups and 260 markers, with an average density of one marker every 4.9 cM. Almost half of the markers (110) deviated from the expected 1:1 segregation ratio (Chi-square p<0.05). These markers were mainly located in chromosomes 2, 5, 7, 9, and 10 (data not shown).

3.4.4. QTL mapping for virus resistance

QTLs for resistance to virus inoculation mapped to chromosomes 1, 2, 3, 6, and

10 (Table 3.3). Since AUDPC and incidence were highly correlated (p < 0.0001) for all of the virus diseases, and we did not see major differences in the patterns of the QTLs between incidence and AUDPC, we just show disease incidence QTLs (Fig. 3.4). The resistance alleles were all derived from the resistant parent Oh1VI. The statistical associations for all QTLs were confirmed using Kruskal-Wallis test and single marker analysis (results not shown). Clusters of virus resistance QTLs were found on chromosomes 2, 3, 6, and 10. The largest cluster of QTLs was located on the short arm of chromosome 6 where QTLs for all six viruses were detected. Resistance QTLs for five

82 viruses mapped to the same or nearby regions of chromosome 3. Clusters of resistance

QTLs exclusive to the rhabdoviruses (MFSV and MMV) and potyviruses (MDMV and

SCMV) were found on chromosomes 2 and 10, respectively (Table 3.3, Fig. 3.4).

Major QTLs for reduced MFSV incidence and AUDPC were detected between markers PZA02418.2 and bnlg1520 on the long arm of chromosome 2. This region explained 60% and 52% of the phenotypic variance for the two scoring methods, respectively. Additionally, a minor QTL for low MFSV incidence and AUDPC was detected between markers PZA03047-12 and PZA00540-3 on the short arm of chromosome 6. This region explained 3% of the variance in incidence and 5% of the variance in AUDPC (Table 3.3).

QTLs for MMV incidence and AUDPC mapped to chromosomes 1, 2, 3 and 6

(Fig. 3.4), and explained between 41% and 46% of the total phenotypic variation. QTL for reduced MMV incidence and AUDPC between PZA02964-7 and bnlg1520 on the long arm of chromosome 2 explained 20% and 32% of the phenotypic variance, respectively. Minor QTLs for MMV incidence mapped to chromosomes 1, 3, and 6 and each explained between 5% and 10% of the phenotypic variance. Similar QTLs for MMV

AUDPC on chromosomes 3 and 6 were identified, but not chromosome 1 (Table 3.3).

For MCDV, QTL for reduced incidence and AUDPC were mapped to chromosomes 2, 3, and 6 (Fig. 3.4). Together, they accounted for 35% of the total phenotypic variance. QTLs on chromosome 2 between markers PHM1511-14 and

PHM3309-8 explained 7% and 4% of the variance for incidence and AUDPC, respectively. QTLs on chromosome 3 for MCDV incidence and AUDPC were located

83 between markers PZA00627-1 and PHM13420-11, and explained 16% and 18% of the total phenotypic variance, respectively. QTLs incidence and AUDPC on the short arm of chromosome 6 explained 12% and 13% of the total phenotypic variance, respectively

(Table 3.3).

QTLs for MDMV and WSMV incidence were located on chromosomes 3, 6, and

10 (Fig. 3.4). A major QTL for reduced MDMV incidence was mapped to the short arm of chromosome 6. This QTL explained 79% of the total phenotypic variance.

Additionally, minor QTLs each explaining 1% of the variance mapped to chromosomes 3 and 10. QTLs for reduced WSMV incidence were mapped to the same or nearby regions as the QTLs for MDMV incidence. Each WSMV QTL explained between 7% and 12% of the total phenotypic variance. QTLs for reduced SCMV incidence mapped to chromosomes 3 and 6 in the same region where QTLs for WSMV and MDMV incidence were located. QTLs for SCMV incidence accounted for 31% of the total phenotypic variance (Table 3.3).

3.4.5. Interaction between QTLs

Significant QTL interactions, confirmed by orthogonal contrasts (p<0.0001), were detected between QTLs on chromosome 3 and 6 for MMV, MCDV, MDMV and SCMV

(Fig. 3.4). In addition, there was a significant interaction (p<0.0001) between the MMV resistance QTLs located on chromosomes 1 and 2. According to variance component analysis, none of the QTL interactions explained a higher percentage of variance than the sum of their single QTL effects, with an exception for SCMV. The interaction between

84 the SCMV-resistance QTL on chromosomes 3 and 6 explained up to 28% more of the total variance than the sum of their individual QTL effects (data not shown).

3.5. Discussion

Some viral diseases in maize are able to reduce yield and jeopardize both food security and industry grain-supply (Ali and Yan, 2012; Bonamico et al., 2012; De-

Oliveira et al., 2004; Gordon et al., 1981; Vasquez and Mora, 2007; Wangai et al., 2012).

Genetic resistance is the most widely applied, reliable, cost-effective, and environmentally friendly approach for controlling viral diseases (Gomez et al., 2009;

Kang et al., 2005; Redinbaugh et al., 2004). The identification of clusters of genes conferring resistance to multiple virus diseases in Oh1V1 suggests that this line offers potential for breeding programs seeking to protect vulnerable areas of the world through improved resistance. For instance, introgressing the QTL regions conferring resistance to multiple virus diseases (i.e. QTL regions on chromosomes 2, 3, 6, and 10) into elite cultivars would allow us to protect maize from existing and emerging viral diseases.

Ongoing research is narrowing down the virus resistance QTL regions to prevent genetic linkage drag.

The genetic architecture of resistance to viral diseases in maize has been less studied than for resistance to fungal or bacterial diseases, and no virus resistance genes in maize have been cloned (Redinbaugh and Pratt, 2009). The first steps for the study of genetic resistance are to determine 1) if the resistance is inherited, 2) the number of genes involved, and 3) their mode of action (Kang et al., 2005). Virus resistance in Oh1VI was

85 transmitted to the F1 and F2 generations. In most cases one, two, or three gene models were sufficient to explain segregation in the F2 generation (Table 3.1), suggesting that a few major loci were needed to impart quantitative resistance. In the F2 generation, the segregation ratios for resistance to the potyviruses MDMV, SCMV, and the tritimovirus

WSMV were consistent with previous studies on diverse germplasm, where two or three genes/QTLs were responsible for the virus resistance (Dussle et al., 2000; Jones et al.,

2007; McMullen et al., 1994; Prazeres De Souza et al., 2008; Wu et al., 2007; Zhang et al., 2003). Discrepancies between the F2 segregation ratios observed for SCMV in 2010 and 2006 may be due to sampling size or genotype by environmental effects on the expression of resistance, as suggested previously (Loesch and Zuber, 1967; McMullen and Louie, 1991). To the best of our knowledge, F2 segregation analysis of resistance to

MCDV, MMV, and MFSV has not been reported before.

Few seedlings in the F1 generation developed symptoms of MFSV, MMV,

MDMV, SCMV and WSMV infection suggesting dominance of the resistance (Table

3.1). Symptoms of MFSV-, MMV-, and MCDV-infection on the few symptomatic F1 seedlings were limited, with an intermediate severity rating that was the average of the parents’ responses. This observation suggests the possibility that two different genetic defense mechanisms may be responsible for the resistance to these viruses in Oh1VI. The most common mechanism could be a dominant gene that suppresses virus multiplication and/or movement within the host, followed by the effect of additive genes that reduce effects of systemic infection. In contrast to previous reports for a number of crop species

(Diaz-Pendon et al., 2004), resistance to potyviruses in Oh1VI was dominant (Table 3.1).

86

Our results corroborate previous studies of maize resistance to these viruses

(Jones et al., 2011; McMullen et al., 1994; Pokorny and Porubova, 2006; Stewart et al.,

2012; Wu et al., 2007). Dominant virus-resistance alleles are often responsible for metabolic host alterations, which include the triggering of a range of defense mechanisms

(Culver and Padmanabhan, 2007; Fraser, 1990; Wise et al., 2007). This kind of virus resistance is referred to as active resistance (Fraser, 1990; Gomez et al., 2009). The majority of described virus resistance genes characterized for dominant alleles in plants fall into the NBS-LRR class of R genes (Gururani et al., 2012). For instance, the dominant gene Tm-22, which confers effective and durable resistance to Tomato mosaic virus in tomato and tobacco (Lanfermeijer et al., 2003; Weber et al., 1993), and Rx, which confers resistance to most Potato virus X (Bendahmane et al., 1999), are genes encoding CC-NBS-LRR proteins (Gomez et al., 2009). However, not all characterized dominant virus resistance genes correspond to this resistance mechanism. The dominant genes RTM1, RTM2, and RTM3 [for Restricted Tobacco etch virus (TEV) Movement] in

Arabidopsis encode a jacalin-like protein, a heat shock protein, and an unknown protein, respectively (Chisholm et al., 2001; Cosson et al., 2010).

On the other hand, Oh1VI resistance to MCDV was mainly additive, since most of the F1 seedlings showed reduced severity disease symptoms close to the midparent value. This confirms previous results where MCDV-resistance conferred by Oh1VI was also additive when crossed with another susceptible inbred line (Jones et al., 2004).

However, the rates of MCDV incidence in our experiments indicated that resistance was mainly recessive since 88% of the F1 seedlings were susceptible to the disease (Table

87

3.1). Recessive resistance comprises around 40% of all known virus resistance in plants

(Diaz-Pendon et al., 2004), and in most cases it has been related to host susceptibility factors that are required for virus replication (Gomez et al., 2009; Gururani et al., 2012).

Many of these host susceptibility factors are eukaryotic translation factors, particularly those in the eIF4E family of translation initiation factors (Robaglia and Caranta, 2006;

Truniger et al., 2008). For example, the nsv locus for susceptibility to Melon necrotic spot virus (MNSV) in melon encodes eIF4E, and any disruption of the gene confers resistance to MNSV (Nieto et al., 2006). Interestingly, the QTL conferring most of the resistance to

MCDV on chromosome 3 (Table 3.3, Fig. 3.4) overlaps the region where maize eIF4E is located according to the Maize Genetics and Genomics Database

(http://www.maizegdb.org).

Resistance QTLs for all six viruses were mapped on the short arm of chromosome

6 in the Oh1VI RIL population, and QTLs for five of the six viruses mapped to the same region on chromosome 3 (Fig 3.4). These regions of the maize genome were previously shown to contain resistance genes for the potyviruses MDMV, SCMV, Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) (Ingvardsen et al., 2010; Jones et al., 2007, Jones et al., 2011, Lu Xiang-Ling et al., 2008, Stewart et al., 2012), the tritimovirus WSMV (McMullen et al., 1994), and the waikavirus MCDV (Jones et al.,

2004). Interestingly, resistance QTLs for the rhabdoviruses MMV and MFSV were also detected in the same regions. A QTL study conducted in Hawaii under natural infestation confirms the MMV resistance loci on chromosome 3 (Ming et al., 1997).

88

The resistance QTLs identified in the Oh1VI RIL population for MDMV and

WSMV on chromosome 10 and MMV on chromosome 1 corroborate previous reports

(Jones et al., 2007; Kyetere et al., 1999; Welz et al., 1998). Maize chromosomes 10 and 1 also contain resistance QTL for other unrelated virus diseases, including Mal del Rio

Cuarto virus (MRCV) and Maize rayado fino virus (MRFV), (Bonamico et al., 2012; Di

Renzo et al., 2004; chapter 4). Resistance to MCDV derived from Oh1VI was previously mapped to the same region on chromosome 10 in an F2 population using a different susceptible parent (Jones et al., 2004).

Major novel QTLs conferring resistance to MMV and MFSV were identified on the long arm of chromosome 2 (Table 3.3, Fig 3.4). These QTLs could be specific against negative sense RNA viruses since all the other tested viruses are positive sense RNA.

Supporting this hypothesis, a QTL conferring resistance to the ambisense Maize stripe virus (MSpV) was previously identified in the same maize genomic region (Dintinger et al., 2005). Another novel QTL for resistance to MCDV was identified on the short arm of chromosome 2 (Fig 3.4). This is the same region where resistance QTLs to Maize streak virus (MSV) and MRCV have also been identified (Kyetere et al., 1999; Martin et al.,

2009; Welz et al., 1998).

Cockerham’s model for QTL epistatic effects (Kao and Zeng, 2002) detected significant interactions (p<0.0001) between all of the resistance QTLs mapped on chromosome 6 and 3 (Fig 3.4). Significant interactions between these resistance loci were reported for MDMV (Jones et al., 2011). Interactions between the resistance loci on chromosomes 6 and 10 were reported for SCMV and MCDV (Jones et al., 2004; Xia et

89 al., 1999). These results suggest that the gene(s) on chromosome 6 interact with other genes either on chromosomes 3 or 10 to confer complete or partial virus resistance, and that the genetic background and/or environment may regulate the role(s) of the genes on chromosomes 3 and 10. The interaction observed between the MMV resistance QTLs located on chromosomes 1 and 2 is noteworthy since major resistance QTLs to MRCV and MSV have been reported in the same region of chromosome 1 (Di Renzo et al., 2004;

Kyetere et al., 1999; Welz et al., 1998).

It is not clear if a single gene or a cluster of genes are responsible for the multiple virus-resistance loci found in some maize chromosomes. About 80% of plant viruses have positive-strand RNA genomes suggesting similar replication, movement or gene regulation strategies (Gomez et al., 2009). A single maize gene targeting one of these common mechanisms may be enough to partially or completely suppress virus replication or movement. For instance, the A. thaliana RTM1 gene restricts long distance movement of Tobacco etch virus (TEV) and other potyviruses by the accumulation of a jacalin-like protein in the phloem tissue (Chisholm et al., 2000; Chisholm et al., 2001). On the other hand, the aforementioned maize genome regions are known to contain clusters of resistance genes for multiple taxonomic groups of pathogens (McMullen and Simcox,

1995; Redinbaugh et al., 2004; Wisser et al., 2006), in addition to defense response gene homologs (Wang et al., 2007) and resistance gene analogues (Xiao et al., 2007). The tendency of resistance genes to cluster is widely known in maize and other plant species

(Friedman and Baker, 2007; Gururani et al., 2012; Lozano et al., 2012; Ribas et al.,

2011), and seems to occur via gene duplication, unequal crossing-over, transposon

90 insertion, and divergence through time by selection and recombination (Friedman and

Baker, 2007; Michelmore and Meyers, 1998). Further characterization and cloning of the maize genes conferring resistance to virus diseases will provide a better understanding of the biological and molecular processes that are important for the development of virus- resistant plants.

In summary, dominant, recessive, additive, and epistatic gene effects were responsible for the multiple-virus resistance observed in the inbred line Oh1VI. In addition, a cluster of QTLs conferring resistance to a diverse set of virus diseases were mapped on chromosomes 3, 6, and 10 at the same regions where virus resistance has been reported before, and a novel major resistance QTL that is effective against negative sense

RNA viruses was identified on chromosome 2.

3.6. Acknowledgments

I thank the following people for contributing to this chapter which was submitted to Theoretical and Applied Genetics: Mark Jones phenotyped the populations with the

Potyviridae and Erick Brenner with MCDV. Margaret Redinbaugh and David Francis advised the research and edited the manuscript. Adriana Tomas (DuPont-Pioneer) and the

Dupont Pioneer Marker Lab for genotyping the RIL population. I thank William Belote

(Dupont, Stine-Haskell Research Center) for providing a P.maidis colony and Jane Todd

(USDA-ARS) for maintaining the insect colonies. I also thank Geoff Parker (USDA-

ARS) for technical assistance with the SSR genotyping and Brayton Orchard (Ohio State

University) for providing the Circos scripts for the QTL graph. I thank the Instituto

91

Nacional Autónomo de Investigaciones Agropecuarias (INIAP), Ecuador for a fellowship to support my Ph.D. study.

92

3.7. References

Ali, F., Yan, J. 2012. Disease Resistance in Maize and the Role of Molecular Breeding in Defending Against Global Threat. J Integr Plant Biology 54:134-151

Balzarini, M., Milligan, S. 2003. Best linear unbiased prediction (BLUP) for genotype performance. In: Kang MS (ed) Handbook of Formulas and Software for Plant Geneticists and Breeders. The Haworth Press, Inc, NY, pp 181-191

Bendahmane, A., Kanyuka, K., Baulcombe, D.C. 1999. The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11:781-791

Bonamico, N.C., Di Renzo, M.A., Ibañez, M.A., Borghi, M.L., Díaz, D.G., Salerno, J.C., Balzarini, M.G. 2012. QTL analysis of resistance to Mal de Río Cuarto disease in maize using recombinant inbred lines. J Agric Sci 150:619

Chisholm, S.T., Parra, M.A., Anderberg, R.J., Carrington, J.C. 2001. Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus. Plant Physiol 127:1667-1675

Chisholm, S.T., Mahajan, S.K., Whitham, S.A., Yamamoto, M.L., Carrington, J.C. 2000. Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus. Proc Natl Acad Sci U S A 97:489-494

Churchill, G.A., Doerge, R.W. 1994. Empirical Threshold Values for Quantitative Trait Mapping. Genetics 138:963-971

Clewer, A.G., Scarisbrick, D.H. 2001. Practical statistics and experimental design for plant and crop science. J. Wiley, Chichester; New York

Coaker, G.L. 2003. Genetic and biochemical characterization of resistance to bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. Dissertation, The Ohio State University

Cosson, P., Sofer, L., Le, Q.H., Leger, V., Schurdi-Levraud. V., Whitham, S.A., Yamamoto. M.L., Gopalan. S., Le Gall, O., Candresse, T., Carrington, J.C., Revers, F. 2010. RTM3, Which Controls Long-Distance Movement of Potyviruses, Is a Member of a New Plant Gene Family Encoding a Meprin and TRAF Homology Domain-Containing Protein. Plant Physiol 154:222-232

Culver, J.N., Padmanabhan, M.S. 2007. Virus-induced disease: Altering host physiology one interaction at a time. Annu Rev Phytopathol 45:221-243

93

De-Oliveira, E., Duarte, A.P., De-Carvalho, R., De-Oliveira, A.C. 2004. Molicutes e virus na cultura do milho no brasil: caracterizacao e factores que afetam sua incidencia. In: De-Oliveira, E., De-Oliveira, C.M. (eds) Doencas en Milho. Molicutes, Virus, Vetores e Mancha por Phaeosphaeria. Embrapa Informacao Tecnologica, Brasilia, pp 17

Di Renzo, M.A., Bonamico, N.C., Diaz, D.G., Ibanez, M.A., Faricelli, M.E., Balzarini, M.G., Salerno, J.C. 2004. Microsatellite markers linked to QTL for resistance to Mal de Rio Cuarto disease in Zea mays L. J Agric Sci 142:289-295

Diaz-Pendon, J., Truniger, V., Nieto, C., Garcia-Mas, J., Bendahmane, A., Aranda, M. 2004. Advances in understanding recessive resistance to plant viruses. Mol Plant Pathol 5:223-233

Dintinger, J., Verger, D., Caiveau, S., Risterucci, A.M., Gilles, J., Chiroleu, F., Courtois, B., Reynaud, B., Hamon, P. 2005. Genetic mapping of maize stripe disease resistance from the Mascarene source. Theor Appl Genet 111:347-359

Dussle, C.M., Melchinger, A.E., Kuntze, L., Stork, A., Luebberstedt, T. 2000. Molecular mapping and gene action of Scm1 and Scm2, two major QTL contributing to SCMV resistance in maize. Plant Breed 119:299-303

Fraser, R.S. 1990. The Genetics of Resistance to Plant Viruses. Cook, R.J. (ed.). Annual Review of Phytopathology, Vol.28.179-200. Annual Reviews Inc.: Palo Alto, California, USA.

Friedman, A.R., Baker, B.J. 2007. The evolution of resistance genes in multi-protein plant resistance systems. Curr Opin Genet Dev 17:493-499

Gomez, P., Rodriguez-Hernandez, A.M., Moury, B., Aranda, M.A. 2009. Genetic resistance for the sustainable control of plant virus diseases: breeding, mechanisms and durability. Eur J Plant Pathol 125:1-22

Gordon, D.T., Bradfute, O.E., Gingery, R.E., Knoke, J.K., Nault, L.R., Scott, G.E. 1981. Introduction: history, geographical distribution, pathogen characteristics and economic importance. In: Gordon, D.T., Knoke, J.K., Scott, G.E. (eds) Virus and Virus-like Disease of Maize in the United States, 247th edn. Southern Cooperative Series Bulletin, Wooster, Ohio, pp 1-12

Gordon, D.T., Thottappilly, G. 2003. Maize and Sorghum. In: Loebenstein G, Thottappilly G (eds) Virus and virus-like diseases of major crops in developing countries. Kluwer Academic Publishers, The Netherlands, pp 295-334

94

Gururani, M.A., Venkatesh, J., Upadhyaya, C.P., Nookaraju, A., Pandey, .S.K, Park, S.W. 2012. Plant disease resistance genes: Current status and future directions. Physiol Mol Plant Pathol 78:51-65

Hull, R. 2002. Matthew's Plant Virology. Academic Press, San Diego

Hunt, R.E., Nault, L.R., Gingery, R.E. 1988. Evidence for Infectivity of Maize Chlorotic Dwarf Virus and for a Helper Component in its Leafhopper Transmission. Phytopathol 78:499-504

Ingvardsen, C.R., Xing, Y., Frei, U.K., Luebberstedt, T. 2010. Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize. Theor Appl Genet 120:1621- 1634

Jansen, R.C., Stam, P. 1994. High-Resolution of Quantitative Traits into Multiple Loci Via Interval Mapping. Genetics 136:1447-1455

Jones, E., Chu, W., Ayele, M., Ho, J., Bruggeman, E., Yourstone, K., Rafalski, A., Smith, O.S., McMullen, M.D., Bezawada, C., Warren, J., Babayev, J., Basu, S., Smith, S. 2009. Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm. Mol Breed 24:165-176

Jones, M.W., Redinbaugh, M.G., Anderson, R.J., Louie, R. 2004. Identification of quantitative trait loci controlling resistance to Maize chlorotic dwarf virus. Theor Appl Genet 110:48-57

Jones, M.W., Redinbaugh, M.G., Louie, R. 2007. The Mdm1 locus and maize resistance to Maize dwarf mosaic virus. Plant Dis 91:185-190

Jones, M.W., Boyd, E.C., Redinbaugh, M.G. 2011. Responses of maize (Zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the Potyviridae. Theor Appl Genet 123:729-740

Kang, B., Yeam, I., Jahn, M.M. 2005. Genetics of plant virus resistance. Annu Rev Phytopathol 43:581-621

Kao, C.H., Zeng, Z.B. 2002. Modeling epistasis of quantitative trait loci using Cockerham's model. Genetics 160:1243-1261

Kosambi, D.D. 1944. The estimation of map distances from recombination values. Ann Eugenics 12:172-175

95

Krzywinski, M.I., Schein, J.E., Birol, I., Connors, J., Gascoyne, R., Horsman, D., Jones, S.J., Marra, M.A. 2009. Circos: An information aesthetic for comparative genomics. Genome Res. doi: 10.1101/gr.092759.109

Kyetere, D.T., Ming, R., McMullen, M.D., Pratt, R.C., Brewbaker, J., Musket, T. 1999. Genetic analysis of tolerance to maize streak virus in maize. Genome 42:20-26

Lanfermeijer, F.C., Dijkhuis, J., Sturre, M.J.G., de Haan, P., Hille, J. 2003. Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-2(2) from Lycopersicon esculentum. Plant Mol Biol 52:1037-1049

Lapierre, H., Signoret, P.A. 2004. Viruses and virus diseases of Poaceae (Gramineae). INRA Ed. Paris, France

Loesch, P.J., Zuber, M.S. 1967. An inheritance study of resistance to Maize dwarf mosaic virus in corn (Zea mays L). Agron J 59:423-&

Louie, R., Knoke, J.K., Reichard, D.L. 1983. Transmission of Maize dwarf mosaic-virus with solid-stream inoculum. Plant Dis 67:1328-1331

Louie, R. 1986. Effects of genotype and inoculation protocols on resistance evaluation of maize to Maize dwarf mosaic virus strains. Phytopathol 76:769-773

Louie, R., Anderson, R.J. 1993. Evaluation of Maize chlorotic dwarf virus resistance in maize with multiple inoculations by Graminella nigrifrons (Homoptera: Cicadellidae). J Econ Entomol 86:1579-1583

Louie, R. 1995. Vascular puncture of maize kernels for the mechanical transmission of maize White line mosaic virus and other viruses of maize. Phytopathol 85:139-143

Louie, R., Abt, J.J., Anderson, R.J., Redinbaugh, M.G., Gordon, D.T. 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family Tombusviridae. Plant Dis 84:1133-1139

Louie, R., Redinbaugh, M.G., Anderson, R.J., Jones, M.W. 2002. Registration of maize germplasm Oh1VI. Crop Sci 42:991-991

Lozano, R., Ponce, O., Ramirez, M., Mostajo, N., Orjeda, G. 2012. Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja. Plos One 7:e34775

Lu Xiang-Ling, Li Xin-Hai, Xie Chuan-Xiao, Hao Zhuan-Fang, Ji Hai-Lian, Shi Li-Yu, Zhang Shi-Huang. 2008. Comparative QTL mapping of resistance to sugarcane mosaic virus in maize based on bioinformatics. Yichuan 30:101-108 96

Martin, T., Franchino, J.A., Kreff, E.D., Procopiuk, A.M., Tomas, A., Luck, S.D., Shu, G.G. 2009. Major QTL conferring resistance of corn to Fijivirus. US2008/012327

McMullen, M.D., Louie, R. 1989. The linkage of molecular markers to a gene controlling the symptom response in maize to Maize dwarf mosaic virus. Mol Plant-Microbe Interact 2:309-314

McMullen, M.D., Louie, R. 1991. Identification of a gene for resistance to Wheat streak mosaic virus in maize. Phytopathol 81:624-627

McMullen, M.D., Louie, R., Simcox, K.D., Jones, M.W. 1994. Three genetic loci control resistance to Wheat streak mosaic virus in the maize inbred Pa405. Molecular plant- microbe interactions : MPMI 7:708-712

McMullen, M.D., Simcox, K.D. 1995. Genomic organization of disease and insect resistance genes in maize. Mol Plant-Microbe Interact 8:811-815

Melchinger, A.E., Kuntze, L., Gumber, R.K., Lubberstedt, T., Fuchs, E. 1998. Genetic basis of resistance to sugarcane mosaic virus in European maize germplasm. Theor Appl Genet 96:1151-1161

Michelmore, R.W., Meyers, B.C. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res 8:1113-1130

Ming, R., Brewbaker, J.L., Pratt, R.C., Musket, T.A., McMullen, M.D. 1997. Molecular mapping of a major gene conferring resistance to Maize mosaic virus. Theor Appl Genet 95:271-275

Nault, L.R., Knoke, J.K. 1981. Maize vectors. In: Knoke, J.K., Gordon, D.T., Scott, G.E. (eds). Virus and Virus-like Diseases of Maize in the United States. South. Coop. Ser. Bull., Wooster, Ohio, pp 77-84

Nieto, C., Morales, M., Orjeda, G., Clepet, C., Monfort, A., Sturbois, B., Puigdomenech, P., Pitrat, M., Caboche, M., Dogimont, C., Garcia-Mas, J., Aranda, M.A., Bendahmane, A. 2006. An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. Plant J 48:452-462

Pokorny, R., Porubova, M. 2006. Heritability of resistance in maize to the Czech isolate of Sugarcane mosaic virus. Cereal Res Commun 34:1081-1086

Prazeres De Souza, I.R., Schuelter, A.R., Guimaraes, C.T., Schuster, I., De Oliveira, E., Redinbaugh, M. 2008. Mapping QTL contributing to SCMV resistance in tropical maize. Hereditas (Lund) 145:167-173

97

Redinbaugh, M.G., Louie, R., Ngwira, P., Edema, R., Gordon, D., Bisaro, D. 2001. Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation. J Virol Methods 98:135-143

Redinbaugh, M.G., Houghton, W., Salomon, R., Creamer, R., Hogenhout, S.A., Gordon, D.T., Ackerman, J., Meulia, T., Seifers, D.L., Abt, J.J., Styer, W.E., Anderson, R.J. 2002. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathol 92:1167- 1174

Redinbaugh, M.G., Gingery, R.E., Jones, M.W. 2004. The genetics of virus resistance in maize (Zea mays L.). Maydica 49:183-190

Redinbaugh, M.G., Pratt, R.C. 2009. Virus Resistance. In: Bennetzen, J.L., Hake, S.C. (eds). Handbook of maize: Its Biology. Springer, New York, pp 251-268

Ribas, A.F., Cenci, A., Combes, M., Etienne, H., Lashermes, P. 2011. Organization and molecular evolution of a disease-resistance gene cluster in coffee trees. BMC Genomics 12:240

Robaglia, C., Caranta, C. 2006. Translation initiation factors: a weak link in plant RNA virus infection. Trends Plant Sci 11:40-45

Shulka, D.D., Wardand, C.W., Brunt, A.A. 1994. The Potyviridae. CAB International, Oxon, UK

Slykhuis, J.T. 1955. Aceria tulipae Keifer (Acarina: Eriophyidae) in relation to spread of Wheat streak mosaic virus. Phytopathol 45:116-128

Stenger, D.C., Hall, J.S., Choi, I., French, R. 1998. Phylogenetic relationships within the family Potyviridae: Wheat streak mosaic virus and Brome streak mosaic virus are not members of the genus . Phytopathol 88:782-787

Stewart, L.R., Md, A.H., Jones, M.W., Redinbaugh, M.G. 2012. Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus. Mol Breed. doi:10.1007/s11032-012-9789-5

Sun, C., Zhang, G., Li, M., Wang, X., Zhang, G., Tian, Y., Wang, Z. 2010. Sequence characterized amplified region markers tightly linked to the dwarf mosaic resistance gene Mdm1 (t) in maize (Zea mays L.). Euphytica 174:219-229

Thottappilly, G., Bosqueperez, N.A., Rossel, H.W. 1993. Viruses and virus diseases of maize in tropical Africa. Plant Pathol 42:494-509

98

Todd, J.C., Hoy, C., Hogenhout, S.A., Ammar, E., Redinbaugh, M.G. 2010. Plant host range and leafhopper transmission of Maize fine streak virus. Phytopathol 100:1138-1145

Truniger, V., Nieto, C., Gonzalez-Ibeas, D., Aranda, M. 2008. Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant. Plant J 56:716-727

Uyemoto, J.K., Bockelman, D.L., Claflin, L.E. 1980. Severe outbreak of corn lethal necrosis disease in Kansas. Plant Dis 64:99-100 van Ooijen, J.W., Boer, M.P., Jansen, R.C., Maliepaard, C. 2002. MapQTL 4.0, Software for the calculation of QTL positions on genetic maps van Ooijen, J.W., Voorrips, R.E. 2001. JoinMap version 3.0, software for the calculation of genetic linkage maps

Vasquez, J., Mora, E. 2007. Incidence of and yield loss caused by Maize rayado fino virus in maize cultivars in Ecuador. Euphytica 153:339-342

Wang, G., Chen, Y., Zhao, J., Li, L., Korban, S.S., Wang, .F, Li, J., Dai, J., Xu, M. 2007. Mapping of defense response gene homologs and their association with resistance loci in maize. J Integr Plant Biology 49:1580-1598

Wangai, A.W., Redinbaugh, M.G., Kinyua, Z.M., Miano, D.W., Leley, P.K., Kasina, M., Mahuku, G., Scheets, K., Jeffers, D. 2012. First reposr of Maize chlorotic mottle virus and Maize lethal necrosis in Kenya. Plant Dis 96:1582-1582

Weber, H., Schultze, S., Pfitzner, A.J.P. 1993. Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome the Tm-2-2 resistance gene in the tomato. J Virol 67:6432-6438

Welz, H.G., Schechert, A., Pernet, A,, Pixley, K.V., Geiger, H.H. 1998. A gene for resistance to the Maize streak virus in the African CIMMYT maize inbred line CML202. Mol Breed 4:147-154

Williams, M.M., Pataky, J.K. 2012. Interactions between maize dwarf mosaic and weed interference on sweet corn. Field Crops Res 128:48-54

Wise, R.P., Moscou, M.J., Bogdanove, A.J., Whitham, S.A. 2007. Transcript profiling in host-pathogen interactions. Annu Rev Phytopathol 45:329-369

Wisser, R.J., Nelson, R.J., Balint-Kurti, P. 2006. The genetic architecture of disease resistance in maize: a synthesis of published studies. Phytopathol 96:120-129 99

Wu, J., Ding, J., Du, Y., Xu, Y., Zhang, X. 2007. Genetic analysis and molecular mapping of two dominant complementary genes determining resistance to Sugarcane mosaic virus in maize. Euphytica 156:355-364

Xia, X.C., Melchinger, A.E., Kuntze, L., Lubberstedt, T. 1999. Quantitative trait loci mapping of resistance to Sugarcane mosaic virus in maize. Phytopathol 89:660-667

Xiao, W., Zhao, J., Fan, S., Li, L., Dai, J., Xu, M. 2007. Mapping of genome-wide resistance gene analogs (RGAs) in maize (Zea mays L.). Theor Appl Genet 115:501-508

Zambrano, J.L., Francis, M.D., Redinbaugh, M.G. 2013. Identification of resistance to Maize rayado fino virus in maize inbred lines. Plant Dis in press

Zhang, S.H., Li, X.H., Wang, Z.H., George, M.L., Jeffers, D., Wang, F.G., Liu, X.D., Li, M.S., Yuan, L.X. 2003. QTL mapping for resistance to SCMV in chinese maize germplasm . Maydica 48:307-312

100

w x y z Location Virus Trait Oh1VI Oh28 F1 F2 F2 ratio Green- MFSV Incidence 0/20 19/19 1/55 20/88 3:1, 13:3 house Severity 1.0 ± 0 5.0 ± 0 3.0 ± 0 4.3 ± 1 MMV Incidence 0/15 19/20 7/60 39/95 9:7 Severity 1.0 ± 0 5.0 ± 0 3.0 ± 0 4.4 ± 1 MCDV Incidence 0/14 16/16 42/48 88/100 13:3 Severity 1.0 ± 0 5.0 ± 0 3.2 ± 1 3.5 ± 1 Field 2010 MDMV Incidence 0/14 85/85 0/10 21/219 2006 0/17 41/42 0/34 18/229 15:1 2010 SCMV Incidence 0/23 63/66 0/16 65/235 3:1, 11:5 2006 0/7 18/19 1/20 50/294 13:3 2010 WSMV Incidence 0/34 68/79 0/26 2/202 63:1 wGreenhouse results are from maize seedlings at 21 days post inoculation. Field results are from maize plants 14 to 21 days post inoculation in summers 2010 and 2006. xMFSV, Maize fine streak virus; MMV, Maize mosaic virus; MCDV, Maize chlorotic mosaic virus; MDMV, Maize dwarf mosaic virus; SCMV, Sugar cane mosaic virus; WSMV, Wheat streak mosaic virus. yIncidence number of symptomatic plants/total number plants. Severity, mean ± one standard deviation of severity ratings for symptomatic plants only (1- 5 scale), where 1 = no symptoms and 5 = severe symptoms. z Possible resistant: susceptible ratios in the F2 determined using a Chi square test (p>0.05).

Table 3.1. Inheritance of maize resistance to six viruses in F1 and F2 generations derived from the resistant inbred line Oh1VI and the susceptible Oh28.

101

Incidence AUDPC Sources MFSV MMV MCDV MDMV SCMV MFSV MMV MCDV Genotype 16.80 12.88 4.13 1351.50 403.57 196.43 375.97 97.73 Replication 0.47 0.07 1.37 33.40 68.42 32.03 11.38 19.56 Residual 7.96 12.10 12.78 289.86 272.56 106.57 170.82 205.15 Total 25.23 25.05 18.28 1674.76 744.55 335.03 558.17 322.44 zMFSV, Maize fine streak virus; MMV, Maize mosaic virus; MCDV, Maize chlorotic mosaic virus; MDMV, Maize dwarf mosaic virus; SCMV, Sugar cane mosaic virus.

Table 3.2. Components of variance for disease incidence and area under the disease progress curve (AUDPC) for a maize recombinant inbred line (RIL) population inoculated with five viruses.z

102

Maize Virus Trait Most LODz Variance Flanking Markers Chr. Dis.y Likely Explai- Position ned (%) cM 1 MMV Incidence 66.6 4.4 5 PHM2177.85 - PHM5098.25 2 MCDV Incidence 21.9 5.1 7 PHM1511.14 - PHM3309.8 AUDPC 21.9 3.2 4 MFSV Incidence 135.3 40.5 60 PZA02418.2 - bnlg1520 AUDPC 135.3 35.9 52 MMV Incidence 135.3 14.4 20 PZA02964.7 - bnlg1520 AUDPC 135.3 22.9 32 3 MMV Incidence 43.3 4.6 6 PZA00627.1 - PHM13420.11 AUDPC 43.3 5.3 6 MCDV Incidence 47.8 12.4 16 PZA00627.1 - PHM13420.11 AUDPC 47.8 13.8 18 SCMV Incidence 47.8 10.4 13 PZA00627.1 - PHM13420.11 WSMV Incidence 47.8 7.0 10 PZA00627.1 - PHM13420.11 MDMV Incidence 52.5 3.4 1 PZA02589.1 - PHM9914.11 6 MDMV Incidence 1.1 93.5 79 PHM15961.13 - PZA03047.12 SCMV Incidence 1.1 13.6 18 PHM15961.13 - PZA03047.12 WSMV Incidence 1.1 8.3 12 PHM15961.13 - PZA03047.12 MCDV Incidence 2.1 9.5 12 PHM15961.13 - PZA00540.3 AUDPC 2.1 10.2 13 MFSV Incidence 3.5 3.2 3 PZA03047.12 - PZA00540.3 AUDPC 3.5 4.4 5 MMV Incidence 3.5 8.2 10 PZA00503.5 - PZA00540.3 AUDPC 3.5 6.6 8 10 WSMV Incidence 39.4 5.4 7 PHM1812.32 - PHM13687.14 MDMV Incidence 43.3 3.3 1 PZA00337.3 - PHM15868.56 yMFSV, Maize fine streak virus; MMV, Maize mosaic virus; MCDV, Maize chlorotic mosaic virus; MDMV, Maize dwarf mosaic virus; SCMV, Sugar cane mosaic virus; WSMV, Wheat streak mosaic virus. zLOD p< 0.01 based on permutation.

Table 3.3. Location and genetic effects of QTLs associated with virus resistance in maize inbred line Oh1VI.

103

Fig. 3.1. Symptom severity scale for: A, Maize mosaic virus; B, Maize fine streak virus; and C, Maize chlorotic dwarf virus. Where, 1 = no disease symptoms, 3 = mild or incomplete symptoms and 5 = severe symptoms.

104

MMV MFSV MCDV

140 140 140

80 80 80

40 40 40

Frequency Frequency Frequency

0 0 0

0 20 40 60 80 0 20 40 60 80 0 20 40 60 80

Disease Incidence (%) Disease Incidence (%) Disease Incidence (%)

MDMV SCMV WSMV

150 150 150

50 50 50

Frequency Frequency Frequency

0 0 0

0 20 40 60 80 0 20 40 60 80 0 20 40 60 80

Disease Incidence (%) Disease Incidence (%) Disease Incidence (%)

Fig. 3.2. Mean distribution of symptom incidence among 256 maize RILs inoculated with Maize mosaic virus (MMV), Maize fine streak virus (MFSV), Maize chlorotic dwarf virus (MCDV) and Wheat streak mosaic virus (WSMV), Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus (SCMV) evaluated between 14 and 21 days post inoculation.

105

MMV MFSV MCDV

140 140 140

100 100 100

60 60 60

Frequency Frequency Frequency

20 20 20

0 0 0

0 10 20 30 40 50 60 0 10 20 30 40 50 0 10 20 30 40 50

AUDPC AUDPC AUDPC

Fig. 3.3. Mean distribution of area under the disease progress curve (AUDPC) scores for 256 maize RILs inoculated with Maize mosaic virus (MMV), Maize fine streak virus (MFSV) and Maize chlorotic dwarf virus (MCDV) evaluated at 21 days post inoculation.

106

Fig. 3.4. Genetic location of maize QTLs conferring resistance to virus diseases. The bars indicate significant LOD scores (p < 0.01) across the Oh1VI x Oh28 genetic map (cM) for: a, Maize chlorotic dwarf virus (MCDV); b, Maize mosaic virus (MMV); c, Maize fine streak virus (MFSV); d, Maize dwarf mosaic virus (MDMV); e, Sugarcane mosaic virus (SCMV); and f, Wheat streak mosaic virus (WSMV). The fine gray circle within the band for each virus indicates the significance threshold for LOD scores. The ribbons link the regions where QTL interactions for some virus diseases were detected (p < 0.0001).

107

CHAPTER 4

QTL MAPPING OF RESISTANCE TO MAIZE RAYADO FINO VIRUS

4.1. Abstract

Maize rayado fino virus (MRFV) is one of the most important virus diseases of maize in regions of Mexico, Central and South America where moderate to severe yield losses have been described. The virus has been reported from the southern United States to northern Argentina where its vector, the leafhopper Dalbulus maidis, is present.

Although resistance to MRFV has been described in maize lines of tropical origin, little was known about genes or QTL conferring resistance to MRFV. In order to identify the location of gene(s) conferring resistance to MRFV, two recombinant inbred line (RIL) mapping populations segregating for MRFV resistance were inoculated using viruliferous leafhoppers, and their responses to virus infection were evaluated in the greenhouse at 7,

14 and 21 days post inoculation. A quantitative trait locus (QTL) explaining up to 23% of the total phenotypic variance was mapped on chromosome 10 in both populations, with similar genetic and physical positions identified in the two populations. The magnitude of the QTL effect and the validation in two independent populations suggests that resistance to MFRV could be transferred into elite lines in order to develop resistant cultivars.

108

4.2. Introduction

Maize rayado fino virus (MRFV) is one of the most important virus diseases of maize in regions of Mexico, Central and South America (De-Oliveira et al., 2004; Kogel et al., 1996; Vandeplas, 2003; Vasquez and Mora, 2007). First described in 1969 in Costa

Rica (Gamez, 1969), MRFV has also been found from the southern United States to northern Argentina (Bradfute et al., 1979; Kogel et al., 1996). MRFV belongs to the genus Marafivirus (family Tymoviridae), and is characterized by isometric virions of approximately 30 nm in diameter, and a single-stranded positive-sense RNA genome

(Leon and Gamez, 1981).

In nature, MRFV is transmitted by the maize leafhopper (Dalbulus maidis,

DeLong & Wolcott) in a persistent manner (Nault et al., 1980), and it is often found in plants co-infected with corn stunt spiroplasma (CSS; Spiroplasma kunkelii) and maize bushy stunt phytoplasma (MBSP; Candidatus Phytoplasma astris subgroup 16SrI-B)

(Vandeplas, 2003). Together, the three pathogens form a disease complex known as

“achaparramiento” or red stunt (Hammond and Bedendo, 2001) that cause severe yield losses in tropical and subtropical regions of the Americas (Carpane, 2007; Nault and

Bradfute, 1979; Silveira et al., 2008).

Hundreds of cultivars, including landraces, hybrids, and inbred lines, have been reported as susceptible to MRFV. Initially, only three tropical accessions were identified as resistant (Bustamante et al., 1998; Espinoza and Gamez, 1980; Nault et al., 1980;

Ramirez Rojas et al., 1988; Toler et al., 1985; Vandeplas, 2003) and most of these accessions were open pollinated cultivars, complicating the use of this germplasm for use

109 in genetic analysis and breeding. However, populations segregating for resistance to

MRFV and suitable for genetic mapping have recently been identified (Zambrano et al.,

2013). The purpose of this study was to map the quantitative trait loci (QTL) associated with resistance to MRFV in these unrelated maize populations. To the best of our knowledge, this is the first report of a QTL conferring resistance to MRFV.

4.3. Materials and methods

4.3.1. Plant material

Two maize RIL populations derived from the MRFV resistant inbred lines Oh1VI

(PI 614734) and Ki11 (Ames 27124) and the disease susceptible lines B73 and Oh28

(NSL 91618) (Toler et al., 1985; Zambrano et al., 2013) were selected to study the genetics of resistance. Seeds of 256 recombinant inbred lines (RILs) were S7 – S9 plants developed from the cross between Oh1VI and Oh28 (Chapter 3). Seed of 193 RILs derived from Ki11 x B73, a sub-population of the nested association maize (NAM) population (McMullen et al., 2009; Yu et al., 2008), were obtained from Michael

McMullen (USDA, ARS, Columbia, MO).

4.3.2. Vector and virus

Dalbulus maidis, originally collected from California, was used as a vector to inoculate plants with the virus. Viruliferous D. maidis were obtained by feeding healthy nymphs on MRFV-infected sweet corn plants (‘Spirit’, Syngenta) for 26 days before being used for inoculation (Nault et al., 1980). A MRFV isolate collected in Texas by

110

Bradfute and others (Bradfute et al., 1979) was used. Virus and vectors are maintained by serial transmission to susceptible maize as described previously (Zambrano et al., 2013).

4.3.3. Disease evaluation

The 256 RILs derived from Oh1VI x Oh28 and the 193 RILs derived from Ki11 x

B73 were inoculated using MRFV infected leafhoppers as described previously

(Zambrano et al., 2013), with a modification in the number of insects employed. Briefly, a six-day old seedling of each RIL was placed into a dacron covered cage containing 700 viruliferous D. maidis (500 adults and 200 2-3-stage nymphs) for an inoculation access period (IAP) of seven days in a growth chamber along with 10 seedlings of the correspondent susceptible parental line. After the IAP, plants were fumigated and transferred to a greenhouse for symptom development. Screening of the populations was replicated three times between autumn 2012 and winter 2013 at The Ohio State

University, in the Plant Pathology Department facilities in Wooster.

Disease incidence was estimated based on the presence (value = 1) or absence

(value = 0) of disease symptoms in the plant 21 days post inoculation (dpi), and disease severity was evaluated at 7, 14 and 21 dpi using the 6-point MRFV severity scale described previously (Zambrano et al., 2013). Briefly, the scale ranged from 0 = no symptoms to 5 = a previously symptomatic plant that died. The area under disease progress curve (AUDPC) was calculated from the severity data at 7, 14, and 21 dpi.

AUDPC is a useful quantitative summary of disease intensity over time (Madden et al.,

111

2007). Limitations imposed per our APHIS permit prevented us from keeping the plants longer than 21 days.

4.3.4. Analysis of phenotypic data

Variance components for disease incidence and AUDPC were estimated for each population by restricted maximum likelihood (REML) using Proc Mixed in SAS software, version 9.2 (SAS Institute Inc., Cary, NC). Best linear unbiased predictors or

BLUPS (Balzarini and Milligan, 2003) for incidence and AUDPC of each RIL were calculated using SAS PROC MIXED in SAS software. The model applied was: y = rep + gen + e; where y was the response variable to a virus inoculation, rep was the replication term, gen represented the individual RIL effect, and e the error term in the model. All effects were considered to be random.

4.3.5. Molecular Data

Genotypic information for the RIL derived from Ki11 x B73 was obtained from

Panzea (http://www.panzea.org/) and a genetic map with 742 polymorphic SNP markers distributed through the 10 maize chromosomes was built using JoinMap, version 3 (van

Ooijen and Voorrips, 2001). The map quality (length of chromosomes and gaps between markers) was assessed in comparison to the NAM map v2009 with 1106 SNP markers

(McMullen et al., 2009). Genotypic and map construction for the Oh1VI x Oh28 RIL population was reported previously (Chapter 3). Briefly, a genetic map containing 260 markers distributed on 10 linkage groups was constructed from the genotypes of 256 RIL

112 derived from a 768 SNP marker multiplex assay (Jones et al., 2009) for the Illumina®

BedArray™ platform (Illumina, Inc., San Diego, CA, USA) and 21 SSR markers.

4.3.6. QTL analysis

For both RIL populations, QTL analysis for MRFV resistance was conducted using composite interval mapping (CIM) in MapQTL, version 4 (van Ooijen et al., 2002).

The significance thresholds of the LOD scores (p < 0.001) were estimated by permutation over each linkage group. CIM mapping results for disease incidences were confirmed by

Kruskal-wallis test based on single marker-trait analysis. BLUPS from MRFV incidence and AUDPC were used for QTL mapping.

4.4. Results

Disease symptoms were evaluated at 7, 14 and 21 dpi. Disease incidence and severity ratings in the susceptible parents Oh28 and B73 (Fig. 4.1) resembled the MRFV disease progress in the RIL populations during the evaluation period (data not shown). At

7 dpi, 81% of the Oh28 and B73 seedlings had disease symptoms. The number of symptomatic plants increased slightly until 21 dpi. At 21 dpi, disease incidence and severity ratings in the susceptible parents (controls) were more than 90% and 3.4, respectively. These results indicated that our virus inoculation method was highly effective with an average of 10% or fewer escapes.

Resistance to MRFV was observed in RILs from both mapping populations.

Disease incidence and AUDPC for the RIL populations showed continuous variation,

113 ranging from highly resistant to completely susceptible in both populations (Fig. 4.2).

Variance components analysis of disease incidence and AUDPC was used to determine the contribution of genotype to the total observed variance for both populations (Table

4.1). The proportion of genetic variance explained based on AUDPC and incidence in the

Oh1VI x Oh28 RIL population was 18% and 12%, respectively. Similarly, the genetic effects for AUDPC and incidence in the Ki11 x B73 RIL population were 19% and 16%, respectively. Variance due to replication effect for both traits and populations was less than 4%. Disease incidence and AUDPC based on severity data were highly correlated (r

= >0.90, p = <0.0001) in both populations (data not shown).

In developing the Ki11 x B73 genetic map for QTL analysis, we observed similar genetic position and distance between markers and substantial co-linearity between the de-novo Ki11 x B73 map constructed for this study and the NAM map (McMullen et al.,

2009); therefore, we used the marker positions from the NAM map since this was available (http://www.panzea.org/). Co-linearity between markers that were present in both genetic maps (i.e. Ki11 x B73 and Oh1VI x Oh28) was also observed (data not shown).

QTLs for reduced MRFV incidence and AUDPC were detected on chromosome

10 for both RIL populations (Table 4.2, Fig. 4.3). Resistance alleles in both populations were derived from the virus resistant parent, either Oh1VI or Ki11. The statistical associations for the QTLs were confirmed by Kruskal-Wallis test using single marker- trait analysis that confirmed highly significant relationships between markers on chromosome 10 and disease incidence (P < 0.01). The peak LOD score in the QTL

114 region suggested that the most likely position of the resistance QTL in the Oh1VI x Oh28

RIL population was 52.6 cM, flanked by markers PHM13687.14 and PHM15868.56, for disease incidence and AUDPC. The most likely position of the QTLs for reduced disease incidence and AUDPC in the Ki11 x B73 RIL population was 57.2 and 61.6 cM, respectively, with markers PZA00647.9 and PZA01456.2 flanking the region. In both populations, these QTLs explained up to 23% of the total phenotypic variation. The physical position according to the flanking markers co-localized to 127 and 136Mb on chromosome 10 on the B73 v2 maize genome map (Table 4.2).

4.5. Discussion

Although restricted to the Americas, MRFV is considered one of the most important virus diseases of maize (Gordon et al., 1981; Gordon and Thottappilly, 2003;

Kogel et al., 1996; Redinbaugh and Pratt, 2009). Severe yield losses, ranging from 10 to

50% in landraces to nearly 100% in inbred lines, have been reported (Gamez, 1983;

Vandeplas, 2003; Vasquez and Mora, 2007) and few sources of resistance have been identified (Bustamante et al., 1998; Toler et al., 1985; Vandeplas, 2003; Zambrano et al.,

2013). The inheritance of resistance to MRFV in the maize inbred line Oh1VI was recently described as dominant and consistent with a one and two gene models

(Zambrano et al., 2013), but the location of the gene (s) conferring resistance remained unknown. In this study we identified MRFV resistance QTL in two maize mapping populations. In both lines, a QTL that mapped on the long arm of chromosome 10 explained up to 23% of the phenotypic variance for resistance (Table 4.2). The QTL

115 spanned a 14 cM region of the Oh1VI x Oh28 genetic map and a 9 cM in the Ki11 x B73 map (Fig. 4.3). The QTL identified in independent and unrelated maize populations overlapped according to the positions of flanking markers on the B73 v2 physical map

(http://www.maizegdb.org/). Further development and analysis of recombinant lines for this region will reduce the length of the QTL and facilitate its use in maize breeding programs.

Interestingly, the position of the QTL conferring resistance to MRFV mapped close to Wsm3, a dominant locus conferring complete resistance to Wheat streak mosaic virus (WSMV) (McMullen et al., 1994) and co-locallized or closely linked with a locus enhancing resistance to Maize dwarf mosaic virus (MDMV), Johnsongrass mosaic virus

(JGMV), and Sorghum mosaic virus (SrMV) (Jones et al., 2011; McMullen et al., 1994;

Stewart et al., 2012). This region of the maize genome also carries mcd2, an additive locus conferring resistance to Maize chlorotic dwarf virus (MCDV) (Jones et al., 2004).

These results support the hypothesis that a cluster of genes conferring resistance to virus diseases is located in this region (McMullen and Simcox, 1995; Redinbaugh et al., 2004;

Wisser et al., 2006). The clustering of disease resistance genes has been reported in several plant species (Friedman and Baker, 2007; Lozano et al., 2012; Ribas et al., 2011), and it seems to be a common phenomenon explained by the presence of multi-gene families of resistance loci that expand through recombination and diverge through time

(Michelmore and Meyers, 1998). Of the cloned dominant R genes conferring resistance to viruses, only the soybean RSV1 gene that confers resistance to Soybean mosaic virus

(SMV) has been located on a cluster of CC-NB-LRRs coding genes (Cournoyer and

116

Dinesh-Kumar, 2011). Alternatively, this region of the maize genome may contain a gene that suppresses common mechanisms required for virus infection (e.g. replication or movement) to confer resistance to several viral diseases as reported for other species. In

Arabidopsis thaliana, the gene RTM1 that confers resistance to Tobacco etch virus

(TEV), Plum pox virus (PPV), and Lettuce mosaic virus (LMV) restricts long distance movement of the viruses through the accumulation of a jacalin-like protein in the sieve elements and phloem tissue (Chisholm et al., 2000; Chisholm et al., 2001; Cosson et al.,

2010). Additional research and cloning of a virus resistance gene in maize is needed to identify the most likely hypothesis.

The use of a QTL identified in bi-parental populations for breeding (e.g. marker- assisted selection) has constraints related with the reproducibility of the QTL effect in subsequent populations (Xu and Crouch, 2008). However, in this case we found the same region of the genome conferred resistance to MRFV in two unrelated populations, suggesting the resistance is functional in diverse genetic backgrounds. Oh1VI is an inbred line originally derived from a maize landrace from the Virgin Islands (Louie et al.,

2002), and Ki11 is an inbred line developed in Thailand by the Thailand National Corn and Sorghum Research Center at the Kasetsart University (http://www.ars-grin.gov/cgi- bin/npgs/acc/display.pl?1645506).

In a previous report on Mendelian segregation of MRFV resistance in a F2 population, we suggested that one or two genes were sufficient to account for the resistance in Oh1VI (Zambrano et al., 2013). The identification of a single major locus conferring resistance to MRFV in RIL populations derived from Oh1VI and Ki11

117 indicates that our original estimate was accurate. However, the relatively low amount of variation explained by the genetic effects in the populations (Table 4.1) might have affected the identification of additional QTL. For instance, regions of chromosome 1 (bin

1.03) and 5 (bin 5.02) of the Oh1VI x Oh28 RIL population that did not reach a significance threshold of P <0.001 were significant at P <0.005 in a permutation test.

The identification of maize inbred lines resistant to MRFV (Zambrano et al.,

2013) and the public availability of genotypic information and genetic maps facilitated the location of the MRFV resistance loci. Validation of the QTL in tropical regions with local MRFV isolates was beyond the scope of this study, but based on similarity of genome sequences of MRFV isolates from the Americas (Chicas et al., 2007), we are optimistic that resistance conferred by the QTL will be effective for MRFV isolates across its range.

4.6. Acknowledgments

I thank M. Redinbaugh and D. Francis for advising and editing this manuscript which is in preparation for submission. I thank M. Jones (USDA-ARS, OARDC) for providing seeds of the Oh1VI x Oh28 population. I also thank Adriana Tomas (DuPont-

Pioneer) and the Dupont Pioneer Marker Lab for genotyping the RIL population. I thank

M. McMullen (USDA-ARS, Columbia, MO) for providing seeds of the Ki11 x B73 RIL population. I am grateful to J. Todd (USDA-ARS, OARDC) for maintaining the Dalbulus maidis colony. I thank the Instituto Nacional Autónomo de Investigaciones

Agropecuarias (INIAP), Ecuador for a fellowship to support my Ph.D. study.

118

4.7. References

Balzarini, M., Milligan, S. 2003. Best linear unbiased prediction (BLUP) for genotype performance. In: Kang, M.S. (ed.). Handbook of Formulas and Software for Plant Geneticists and Breeders. The Haworth Press, Inc, NY, pp 181-191

Bradfute, O.E., Toler, R.W., Boothroyd, C.W., Robertson, D.C., Nault, L.R., Gordon, D.T. 1979. Identification of Maize rayado fino virus in the United States. Plant Dis 64:50-53

Bustamante, P.I., Ramirez, P., Hammond, R. 1998. Evaluation of maize germplasm for resistance to Maize rayado fino virus. Plant Dis 82:50-56

Carpane, P.D. 2007. Host resistance and diversity of Spiroplasma kunkelii as components of corn stunt disease. Dissertation, Oklahoma State University.

Chisholm, S.T., Parra, M.A., Anderberg, R.J., Carrington, J.C. 2001. Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of Tobacco etch virus. Plant Physiol 127:1667-1675

Chisholm, S.T., Mahajan, S.K., Whitham, S.A., Yamamoto, M.L., Carrington, J.C. 2000. Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of Tobacco etch virus. Proc Natl Acad Sci U S A 97:489-494

Cosson, P., Sofer, L., Le, Q.H., Leger, V., Schurdi-Levraud, V., Whitham, S.A. , Yamamoto, M.L., Gopalan, S., Le Gall, O., Candresse, T., Carrington, J.C., and F. Revers. 2010. RTM3, which controls long-distance movement of potyviruses, is a member of a new plant gene family encoding a meprin and TRAF homology domain- containing protein. Plant Physiol. 154:222-232.

Cournoyer, P., Dinesh-Kumar, S.P. 2011. NB-LRR Immune Receptors in Plant Virus Defence. In Caranta, C., Aranda, M. A., Tepfer, M., and Lopez-Moya, J.J. (eds.) Recent advances in plant virology. Caister Academic Press, Norfolk, UK. pp 149-176

De-Oliveira, E., Duarte, A.P., De-Carvalho, R., De-Oliveira, A.C. 2004. Molicutes e virus na cultura do milho no brasil: caracterizacao e factores que afetam sua incidencia. In: De-Oliveira, E., De-Oliveira, C.M. (eds.). Doencas en Milho. Molicutes, Virus, Vetores e Mancha por Phaeosphaeria. Embrapa Informacao Tecnologica, Brasilia, pp 17- 34

Espinoza, A.M., Gamez, R. .980) La ultraestructura de la superficie foliar de cultivares de maiz infectados con el virus del rayado fino. Turrialba 30:413-420

119

Friedman, A.R., Baker, B.J. 2007. The evolution of resistance genes in multi-protein plant resistance systems. Curr Opin Genet Dev 17:493-499

Gamez, R. 1983. The ecology of Maize rayado fino virus in the American tropics. In: Plumb RT, Thresh JM (eds) Plant Virus Epidemiology. Blackwell Scientific Publications, Oxford, pp 268-274

Gamez, R. 1969. A new leafhopper-borne virus of corn in Central America. Plant disease reporter 929-932

Gordon, D.T., Thottappilly, G. 2003. Maize and Sorghum. In: Loebenstein, G., Thottappilly, G. (eds.). Virus and virus-like diseases of major crops in developing countries. Kluwer Academic Publishers, The Netherlands, pp 295-334

Gordon, D.T., Bradfute, O.E., Gingery, R.E., Knoke, J.K., Nault, L.R., Scott, G.E. 1981. Introduction: history, geographical distribution, pathogen characteristics and economic importance. In: Gordon, D.T., Knoke, J.K., Scott, G.E. (eds.). Virus and Virus-like Disease of Maize in the United States, 247th edn. Southern Cooperative Series Bulletin, Wooster, Ohio, pp 1-12

Hammond, R.W., Bedendo, I.P. 2001. Role of Maize rayado fino virus in the etiology of "red stunt" disease in Brazil. Plant Dis 85:99-99

Jones, E., Chu, W., Ayele, M., Ho, J., Bruggeman, E., Yourstone, K., Rafalski, A., Smith, O.S., McMullen, M.D., Bezawada, C., Warren, J., Babayev, J., Basu, S., Smith, S. 2009. Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm. Mol Breed 24:165-176

Jones, M.W., Redinbaugh, M.G., Anderson, R.J., Louie, R. 2004. Identification of quantitative trait loci controlling resistance to Maize chlorotic dwarf virus. Theor Appl Genet 110:48-57

Jones, M.W., Boyd, E.C., Redinbaugh, M.G. 2011. Responses of maize (Zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the Potyviridae. Theor Appl Genet 123:729-740

Kogel, R., Ramirez, P., Hammond, R.W. 1996. Incidence and geographic distribution of Maize rayado fino virus (MRFV) in Latin America. Plant Dis 80:679-683

Leon, P., Gamez, R. 1981. Some physicochemical properties of Maize rayado fino virus. J Gen Virol 16:67-75

Louie, R., Redinbaugh, M.G., Anderson, R.J., Jones, M.W. 2002. Registration of maize germplasm Oh1VI. Crop Sci 42:991-991 120

Lozano, R., Ponce, O., Ramirez, M., Mostajo, N., Orjeda, G. 2012. Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja. Plos One 7:e34775

Madden, L.V., Hughes, G., van den Bosch, F. 2007. The study of plant disease epidemics. American Phytopathological Society, St. Paul

McMullen, M.D., Simcox, K.D. 1995. Genomic organization of disease and insect resistance genes in maize. Mol Plant-Microbe Interact 8:811-815

McMullen, M.D., Louie, R., Simcox, K.D., Jones, M.W. 1994. Three genetic loci control resistance to Wheat streak mosaic virus in the maize inbred Pa405. Molecular plant- microbe interactions : MPMI 7:708-712

McMullen, M.D., Kresovich, S., Villeda, H.S., Bradbury, P., Li, H., Sun, Q., Flint- Garcia, S., Thornsberry, J., Acharya, C., Bottoms, C., Brown, P., Browne, C., Eller, M., Guill, K., Harjes, C., Kroon, D., Lepak, N., Mitchell, S.E., Peterson, B., Pressoir, G., Romero, S., Rosas, M.O., Salvo, S., Yates, H., Hanson, M., Jones, E., Smith, S., Glaubitz, J.C., Goodman, M., Ware, D., Holland, J.B., Buckler, E.S. 2009. Genetic properties of the maize nested association mapping population. Science 325:737-740

Michelmore, R.W., Meyers, B.C. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res 8:1113-1130

Nault, L.R., Bradfute, O.E. 1979. Corn stunt: involvement of a complex of leafhopper- borne pathogens. In: Maramorosch, K., Harris, K.F. (eds.). Leafhopper vectors and plant disease agents. Academic Press, New York, pp 561-586

Nault, L.R., Gordon, D.T., Gingery, R.E. 1980. Leafhopper transmission and host range of Maize rayado fino virus. Phytopathol 70:709-712

Ramirez-Rojas, S., Romero-Rosales, F., Jeffers, D., Martinez-Garza, A., Mejia-Andrade, H. 1988. Reacción de ocho variedades de maíz al virus del rayado fino en Chapingo, México. Agricultura Tecnica en Mexico 24:11-18

Redinbaugh, M.G., Gingery, R.E., Jones, M.W. 2004. The genetics of virus resistance in maize (Zea mays L.). Maydica 49:183-190

Redinbaugh, M.G., Pratt, R.C. 2009. Virus Resistance. In: Bennetzen, J.L., Hake, S.C. (eds.). Handbook of maize: Its Biology. Springer, New York, pp 251-268

Ribas, A.F., Cenci, A., Combes, M., Etienne, H., Lashermes, P. 2011. Organization and molecular evolution of a disease-resistance gene cluster in coffee trees. BMC Genomics 12:240 121

Silveira, F.T., Moro, J.R., da Silva, H.P., de Oliveira, J.A., Perecin, D.O. 2008. Inheritance of the resistance to corn stunt. Pesquisa agropecuária brasileira. 43:1717-1724

Stewart, L.R., Md, A.H., Jones, M.W., Redinbaugh, M.G. 2012. Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus. Mol Breed doi:10.1007/s11032-012-9789-5

Toler, R.W., Harris, K.F., Bockholt,. AJ., Skinner, G. 1985. Reactions of maize (Zea mays) accessions to Maize rayado fino virus. Plant Dis 69:56-57 van Ooijen, J.W., Voorrips, R.E. 2001. JoinMap version 3.0, software for the calculation of genetic linkage maps. Plant Research International, Wageningen, the Netherlands. van Ooijen, J.W., Boer, M.P., Jansen, R.C., Maliepaard, C. 2002. MapQTL 4.0, Software for the calculation of QTL positions on genetic maps. Plant Research International, Wageningen, the Netherlands.

Vandeplas, A. 2003. Evaluation of sixty highland elite maize genotypes for resistance to Maize rayado fino virus. Dissertation, The Katholieke Universiteit Leuven

Vasquez, J., Mora, E. 2007. Incidence of and yield loss caused by Maize rayado fino virus in maize cultivars in Ecuador. Euphytica 153:339-342

Wisser, R.J., Nelson, R.J., Balint-Kurti, P. 2006. The genetic architecture of disease resistance in maize: A synthesis of published studies. Phytopathol 96:120-129

Xu, Y., Crouch, J.H. 2008. Marker-assisted selection in plant breeding: From publications to practice. Crop Sci 48:391-407

Yu, J., Buckler, E.S., McMullen, M.D., Holland, J.B. 2008. Genetic design and statistical power of nested association mapping in maize. Genetics 178:539-511

Zambrano, J.L. 2013. Genetic architecture of resistance to phylogenetically diverse viruses in maize. Dissertation, The Ohio State University

Zambrano, J.L., Francis, M..D, Redinbaugh, M.G. 2013. Identification of resistance to Maize rayado fino virus in maize inbred lines. Plant Dis in press

122

Oh1VI x Oh28 Ki11 x B73 Sources AUDPC Incidence AUDPC Incidence Genotype 78.42 0.03 83.72 0.04 Replication 0.48 0.00 13.86 0.01 Residual 350.00 0.22 335.25 0.20

Table 4.1. Components of variance for disease incidence and area under the disease progress curve in two maize recombinant inbred line populations inoculated with Maize rayado fino virus.

123

RIL / Trait Most LODa Variance Flanking Markers Physical Maize likely Explained position of Bin position (%) QTLb (cM) Oh1VI x Oh28 10.04 Incidence 52.6 9.2 18 PHM13687.14 - 117,9Mb - AUDPC 52.6 12.2 23 PHM15868.56 137,5M

Ki11 x B73 10.04 Incidence 57.2 9.4 21 PZA00647.9 - 127,5Mb - AUDPC 61.6 10.9 23 PZA01456.2 136,9Mb

aLOD P< 0.001 based on permutation. bRange in pair of bases of the physical position of the QTLs estimated from the flanking markers on the B73 physical map from the Maize Genetic and Genomic Database (http://www.maizegdb.org/.)

Table 4.2. Location and genetic effects of QTLs associated with resistance to Maize rayado fino virus in two recombinant inbred line (RIL) populations of maize.

124

100

A

90

80

70 Disease Disease incidence(%)

60 7 dpi 14 dpi 21 dpi

B73 Oh28

5 B

4

5) 5) -

3

2

Disease Disease severity (0 1

0 7 dpi 14 dpi 21 dpi

B73 Oh28

Fig. 4.1. Maize rayado fino virus disease progress in susceptible controls and parents of the maize RIL populations Ki11 x B73 and Oh1VI x Oh28 at 7, 14, and 21 days post inoculation. Data presented are the means ± s.d. for three replications with 10 plants per replication. A, Incidence and B, Mean severity rating of symptomatic plants using a rating scale of 0 to 5, where 0 = no disease symptoms and 5 = dead.

125

A

50 50

20 20

Frequency Frequency

0 0

0 20 50 0.0 0.6

AUDPC Incidence

B

50 50

20 20

Frequency Frequency

0 0

0 30 60 0.0 0.6

AUDPC Incidence

Fig. 4.2. Means for AUDPC and incidence of Maize rayado fino virus at 21 days post inoculation in A) 256 maize RIL population derived from Oh1VI x Oh28, and B) 193 maize RIL population derived from Ki11 x B73.

126

Chromosome_10

PZA02554.1 PZA02221.20 PZA01313.2 PHM3765.7 PZA01451.1 PZA00463.3 0 PZA02554.1 PZA02961.6 PZA00079.1 5 PZA02853.11 10 PZD00033.3 PZA03491.1 15 PZA01597.1 PZA00933.3 20 PHM2828.83 PZA01677.1 PHM5740.9 PZA02941.7 25 PZA01451.1 PZA01877.2 PHM15331.16 PHM12990.15 30 PZA02961.6 PZA00400.3 35 PHM3922.32 PHM12625.18 PHM1812.32 PHM18195.6 40 PHM1155.14 PZA00444.1 PZA00337.3 PZA01292.1 45 PHM13687.14 PZA01919.2 PZA00647.9 PZA02128.3 50 . PHM4341.42 PZA01089.1 55 umc1477 . PHM13687.14 60 PZA02219.2 PHM15868.56 PZA01141.1 65 bnlg1028 PZA03196.1 PZA00866.2 70 PZA00647.9 bnlg2190 PZA01456.2 75 PHM15868.56 PZA01995.2 80 PZA00130.9 PZA03604.1 PZA02969.9 85 PZA03605.1 PZA00007.1 PZA03606.1 90 PZA03607.1 PZA00130.9 95 PZA02969.9 PZA02049.1 100 PHM5435.25 PZA01073.1 105 PHM3736.11 PZA02167.2 110 PZA02578.1 PZA00062.4 PHM10750.26 115 PHM1506.23

Fig. 4.3. Location (cM) of a quantitative trait loci (QTL) for resistance to Maize rayado fino virus on chromosome 10 of maize estimated from the Oh1VI x Oh28 RIL population (left) and Ki11 x B73 RIL population (right). Horizontal dot lines denote markers present in both genetic maps. Hatched and black blocks indicates the genome region covered by the QTL at P < 0.001 based on permutations in the Oh1VI xOh28 and Ki11 x B73 maps, respectively.

127

CHAPTER 5

IDENTIFICATION OF A MAJOR QUANTITATIVE TRAIT LOCUS

CONTROLLING RESISTANCE TO MAIZE NECROTIC STREAK VIRUS IN

MAIZE USING A SELECTIVE MAPPING STRATEGY

5.1. Abstract

Maize necrotic streak virus (MNeSV) was first identified in the United States, and it is closely related to viruses that cause important diseases in regions of Africa, Europe, and South America. A major quantitative trait locus conferring resistance to MNeSV was mapped to the long arm of chromosome 10 using a set of informative recombinant inbred lines selected from two larger maize mapping populations. MNeSV cannot be transmitted by leaf rub-inoculation, and no vector has been identified. Vascular puncture inoculation

(VPI) was used to test the responses of recombinant inbred lines (RIL) derived from the cross between the virus resistance maize line Oh1VI and two susceptible inbred lines

Oh28 and Va35 to the virus. Maize kernels were inoculated in the laboratory and planted in the greenhouse for symptom development and evaluation. Quantitative trait loci (QTL) mapping was conducted independently for each population using composite interval mapping (CIM) and single marker regression (SMR). CIM identified a major QTL

128 explaining up to 46% of the phenotypic variance on maize chromosome 10, bin 10.04, in

Oh1VI. These results were validated in an Oh1VI x Va35 RIL population using SMR, which identified linked markers on chromosome 10 in bins 10.04, 10.05 and 10.06. This

QTL explained up to 70% of the phenotypic variance. Analysis of F1 hybrids suggested that resistance to MNeSV was mainly recessive or additive. These results indicate that

VPI is a useful tool for characterizing resistance in mapping populations. The identification of a major locus conferring resistance to MNeSV could allow us to respond quickly and appropriately to any future outbreak of the disease and provides a better understanding of the chromosomal location and distribution of genes conferring resistance to virus diseases in maize.

5.2. Introduction

Maize necrotic streak virus (MNeSV) was discovered in 2000 in a maize leaf sample from Arizona thought to be infected with Maize chlorotic dwarf virus (MCDV)

(Louie et al., 2000). MNeSV is a member of the Tombusviridae, and is a monopartite single-stranded positive sense RNA virus that encodes five open reading frames (ORF).

MNeSV virions are isometric, and approximately 32 nm in diameter (Lapierre and

Signoret, 2004; Louie et al., 2000). Viruses that cause important diseases are related to

MNeSV, including Maize chlorotic mottle virus (MCMV) and Maize white line mosaic virus (MWLMV). These viruses can cause significant yield reduction in maize in several parts of the world, including the United States, Peru, Italy and Kenya (Castillo and

Hebert, 1974; Lapierre and Signoret, 2004; Redinbaugh and Pratt, 2009; Uyemoto et al.,

129

1980; Wangai et al., 2012). MNeSV symptoms on susceptible maize are severe pale green, yellow, or cream-colored spots and streaks measuring 1 to 2 mm on emerging leaves. Subsequent leaves develop large chlorotic bands that later fuse together and become translucent and necrotic around the edges (Louie et al., 2000). The mechanism of

MNeSV transmission under natural field conditions is still unknown.

MNeSV can be easily transmitted mechanically using vascular puncture inoculation (VPI) (Louie, 1995; Louie et al., 2000). Developed by Louie (1995), VPI is an inoculation technique which has been used for successful mechanical transmission of most major maize viruses (Changa, 1998; Louie, 1995; Louie et al., 2000; Louie and Abt,

2004; Louie et al., 2006; Madriz-Ordenana et al., 2000; Redinbaugh et al., 2002; Weiland and Edwards, 2011). Using minute pins attached to an engraving tool, virus inoculum is driven into the scutellar tissue alongside the embryo of germinating seeds, introducing virus particles into embryonic or vascular cells (Louie and Abt, 2004). VPI has been successfully used to transmit viruses without a vector, and even without knowledge of the identity of its vector (Louie et al., 2000; Redinbaugh et al., 2001).

In genetic analysis of virus resistance, a large number of lines are usually inoculated and their responses evaluated. Selective mapping has been used in QTL studies to reduce the number of individuals that need to be evaluated (Howad et al., 2005;

Simon et al., 2008; Zhang et al., 2007). In selective mapping, lines with more desirable distribution of crossovers can be selected from a larger population (Brown et al., 2000;

Vision et al., 2000; Xu et al., 2005). This mapping technique was validated in maize using data from Jones and co-workers (Jones et al., 2004). In a post-hoc analysis, the

130 location and number of virus resistance QTLs detected was not affected by using a set of

113 individuals selected from the entire population of 314 F2 plants (Zambrano, 2012).

However, reducing the number of lines also reduced the power to detect minor QTLs explaining 6% or less of the phenotypic variance (Simon et al., 2008; Zambrano, 2012).

Selective mapping in conjunction with VPI offers a viable strategy to balance the need to reduce variability through increased replication, while reducing the number of lines to be inoculated.

Resistance to MNeSV in maize was identified in tropical germplasm (Louie et al.,

2000), but the action and location of genes conferring resistance to MNeSV or any other virus in the Tombusviridae family remain unknown. Our goal was to determine the mode of inheritance and location of genes conferring resistance to MNeSV using F1, F2 and informative recombinant inbred lines (RIL) selected from maize mapping populations derived from the virus resistant line, Oh1VI. The inbred line Oh1VI (PI 614734) is a flint corn derived from a Virgin Island population (Louie et al., 2002) that(Louie et al., 2002) is highly resistant to at least nine (MDMV, SCMV, WSMV, HPV, MCDV, MFSV,

MMV, MRFV, MNeSV) phylogenetically distinct viruses, including MNeSV (Jones et al., 2004; Louie et al., 2000; Redinbaugh et al., 2002; Zambrano et al., 2013).

5.3. Materials and methods

5.3.1. Maize germplasm

Oh1VI was crossed with the virus-susceptible lines Oh28 and Va35 (Louie et al.,

2000) to generate F1, F2 and RIL populations. Oh1VI was first crossed with Va35 in the

131 summer of 1995. Then, the F1 plants were selfed to advance to the F2 generation in the winter of 1996 in a greenhouse. Through 1997, 310 F2 ears were generated. Seeds of the

F2 plants were planted ear-to-row and successively self-pollinated. Plants were advanced every year in Wooster, OH and by 2009, 305 RILs were self pollinated between nine to eleven times without selection. The lines were maintained by the Corn, Soybean and

Wheat Quality Research Unit (CSWQRU) at the Ohio Agricultural Research and

Development Center (OARDC), Wooster, OH, USA. The development of the Oh1VI x

Oh28 RILs and progenies were reported previously (Chapter 3).

5.3.2. Virus isolate and inoculum

A MNeSV isolate collected near Cochise, AZ (Louie et al., 2000) was maintained by serial transmission into the susceptible sweet corn hybrid ‘Spirit’ (Syngenta) using vascular puncture inoculation (VPI) (Louie, 1995; Redinbaugh et al., 2001). Inoculum extracts for pathogen transmission were prepared by grinding symptomatic leaf tissue in four volumes of 10 mM potassium phosphate, pH 7 as described (Louie et al., 2000).

5.3.3. Virus inoculations

Seed were inoculated with MNeSV using VPI to determine the inherence of the resistance. Kernels were disinfected in a 30% bleach solution for three minutes and then, they were placed on paper towels (Kleenex, Kimberly Clark, Roswell, GA) over night for drying. Next, the kernels were sorted into groups of 15 in sterile 100 mm x 15 mm polystyrene Petri dishes (Fisher Scientific Inc., Pittsburgh, PA) containing 50 ml of

132 distilled de-ionized sterile water at 30oC for 2.5 hours. The water was removed from the

Petri dishes and five sterile 9 cm filter papers (Whatman 4, Whatman International Ltda.,

Maidstone, England) were placed in each dish. The filter papers were soaked with 5 ml of autoclaved water, and seed was redistributed in dishes with the embryo facing up for inoculation using an engraving tool as previously described (Louie et al., 2000; Louie and

Abt, 2004). Inoculations were done on 195 F2 and 90 F1 kernels of the Oh1VI x Oh28 cross, and 60 F1 and 60 F2 kernels of the Oh1VI x Va35 cross. At least 60 kernels of the resistant and the susceptible parents were inoculated and used as controls (Table 5.1).

Inoculations were conducted by populations at two trials. Briefly, VPI was carried out using five 0.20 mm diameter insect pins (Austerlitz insect pins, Carolina Biological,

Burlington, NC) with an electric engraver (Ideal Industries, Inc., Sycamore, IL). An inoculum droplet (3 µl) was placed on the pericarp covering the scutellum. The inoculum was then introduced into each seed by pressing the vibrating pins around 1 mm into the kernel two times about 1 mm from the embryo axis at a 45o angle. After VPI, kernels were incubated at 30o C for two days. Then, kernels from each Petri dish were planted into two 100 mm plastic pots (Kord Products, Toronto, Canada) containing autoclaved soil and placed in a greenhouse set at 25oC and 18oC (day and night, respectively) for symptom development. Natural light was supplemented with 400W high-pressure sodium lights (P. L. Light System, Beamsville, ON, Canada) to obtain a 12 h light period. The screening of Oh1VI x Oh28 progenies was conducted during fall and winter of 2011-

2012, and for Oh1VI x Va35 progenies in the summer of 2000.

133

5.3.4. Evaluation of disease symptoms

Disease incidence and severity for Oh1VI x Oh28 progenies were recorded at 9,

16, and 23 days post inoculation (dpi), and for Oh1VI x Va35 progenies at 21 dpi.

USDA, APHIS permit conditions prevented longer evaluation of symptom development.

Incidence was estimated as the percentage of symptomatic plants/total number of germinated seeds, and severity was evaluated on the two uppermost open leaves of individual plants using a 1 to 5 scale, where 1 = no symptoms, 2 = mild symptoms, 3 = intermediate symptoms, 4 = moderately severe symptoms, and 5 = severe disease symptoms (Fig. 5.1). The area under disease progress curve (AUDPC) was calculated from the severity data at all three time points (Madden et al., 2007).

Phenotypic data for F1 and F2 plants were analyzed relative to parents in order to assess the degree of dominance associated with resistance (Wu et al., 2007). A Chi square test (Clewer and Scarisbrick, 2001) was conducted to assess 3:1, 15:1, 9:7, 11:5, 13:3, and 63:1 segregation ratios for diseases incidence in the F2 generation. For this analysis, seedlings that showed any disease symptom (severity scale ≥ 2) were rated as susceptible.

5.3.5. Selective mapping and phenotyping of RIL populations

Selective mapping (Brown et al., 2000) was used to identify the most informative recombinant lines from a 256 RIL population derived from the Oh1VI x Oh28 cross.

Genotypic data from 28 polymorphic SSR markers distributed across the maize genome was conducted as described previously (Jones et al., 2004) for the entire RIL population, and used to construct a framework map for selection using MapPop 1.0 (Brown and

134

Vision, 2000). The ‘samplemax’ command and default parameters were used. The analysis was performed three times and lines selected in at least two samples were used for QTL mapping. Markers and their positions were obtained from the Maize Genetics and Genomics Database (http://www.maizegdb.org). Phenotypic evaluation was carried out on selected lines that were inoculated and evaluated for symptom development as described above using an incomplete block design with three independent replications of fifteen kernels per replication. Thirty kernels of the susceptible parent Oh28 were inoculated and used as a control.

A subset of lines from a second RIL population developed from Oh1VI x Va35 was selected. The original Oh1V1 x Va35 RIL consisted of 305 lines, and tested lines were selected based on the presence of Oh1VI alleles for SSR markers in one or more of three main genomic regions: 3.04 – 3.05 (markers: umc1223, umc2263, umc2002), 6.00 –

6.01(umc1753, bnlg1867), and 10.04 - 10.05 (umc1930, bnlg1250, umc2112). These regions were selected based on their linkage with potyvirus and MCDV resistance loci

(Jones et al., 2011; McMullen et al., 1994; Redinbaugh and Pratt, 2009). DNA extraction and SSR genotyping of the RILs were conducted as reported previously (Jones et al.,

2004). The selected lines were homozygous at each of the tested loci. Ten kernels of each selected line were inoculated and evaluated as described above using an incomplete block design with three independent replications.

135

5.3.6. Analysis of RIL phenotypic data

Variance components for disease incidence and AUDPC based on severity were estimated for each RIL population by restricted maximum likelihood (REML) using

PROC MIX in SAS software, version 9.2 (SAS Institute Inc., Cary, NC). Best linear unbiased predictors or BLUPS (Balzarini and Milligan, 2003) for disease incidence and

AUDPC of each RIL were estimated using the model: yij = µ + geni + repj + eij; where yij was the phenotypic response to virus inoculation for the ith genotype in the jth replication, geni represented the individual effect of the ith line, repj was the effect of the jth replication, and eij the residual error term in the model. All effects were considered to be random. Pearson correlation analysis was used to assess the relationship between disease incidence and AUDPC in pair-wise comparisons using PROC CORR in SAS.

5.3.7. QTL analysis

Composite interval mapping (CIM) (Gao Huijiang and Yang Runqing, 2006) was conducted to identify QTLs associated with resistance to MNeSV in the Oh1VI x Oh28

RIL population using MapQTL, version 4 (van Ooijen et al., 2002). The significance threshold of the LOD score (p < 0.001) was determined by permutation over each linkage group. BLUPS estimated from the incidence and AUDPC data were used as the phenotypic variable for the QTL analysis. Genotypic information and a genetic map with

260 markers for the Oh1VI x Oh28 RIL population were reported previously (Chapter 3).

Briefly, the genotyping was conducted using a 768 marker multiplex assay for the

Illumina® BedArray™ platform (Illumina, Inc., San Diego, CA, USA), and a genetic

136 map that contained the informative (polymorphic) SSRs and SNP markers on10 linkage groups was built.

Single marker regression (Edwards et al., 1987) using PROC GLM in SAS software, version 9.2 (SAS Institute Inc., Cary, NC) was used to identify QTL associated with resistance to MNeSV in the selected Oh1VI x Va35 RIL population. The same genotypic data used for the selection of the lines was used for the analysis. The selected lines were sorted into genotypic classes for each of eight marker loci described above and

F tests (Clewer and Scarisbrick, 2001) were used to determine if significant differences (p

< 0.001) existed for disease incidence and AUDPC BLUPS between the classes. We did not use CIM in this population, because of the low genetic coverage provided by the eight markers.

5.4. Results

5.4.1. Inheritance of the resistance

MNeSV disease symptoms were observed in Oh1VI, Oh28, and their F1 and F2 progenies at 9 dpi (Fig. 5.2). Disease incidence increased significantly from 9 to 23 dpi in the virus susceptible Oh28 and the F1 and F2 progenies. Disease incidence in the virus resistant Oh1VI did not increase after 9 dpi. Between 9 and 23 dpi, disease severity in symptomatic Oh28 plants increased from 2.9 to 4.8 (intermediate to severe disease symptoms), while F1 and F2 progenies retained intermediate disease symptoms (Fig. 5.3).

The severity of symptoms in those Oh1VI plants that developed symptoms was moderate during the entire evaluation period. We focused our analysis at 23 dpi, since this was the

137 time when the highest disease incidence and severity were observed in the susceptible parent. Because we were limited to doing these experiments in greenhouses, longer evaluation periods were not possible.

In the screening of Oh1VI x Oh28 F1 and F2 progenies (Table 5.1), Oh1VI had a disease incidence of 12% and a severity score of 2.4. Oh28 was susceptible to MNeSV with 81% disease incidence and a mean severity score of 4.8. For F1 and F2 progenies,

84% and 85% developed disease symptoms, respectively. Based on symptom severity scores, the F1 and F2 progenies had an intermediate reaction to MNeSV with mean severity ratings of 3.2 and 3.4, respectively. Segregation of resistance in F2 seedlings was consistent with a 13:3 susceptible: resistant ratio (p = 0.318), but it differed from 3:1,

15:1, 9:7, 11:5, and 63:1 segregation ratios (p = < 0.05). In the Oh1VI x Va35 screening,

48% and 100% of the Oh1VI and Va35 seedlings, respectively, developed some disease symptoms. The mean severity scores for Oh1VI and Oh28 were 2.1 and 4.2, respectively.

F1 seedlings had 47% disease incidence and a mean severity score of 4.3, and F2 seedlings had 79% incidence with a mean severity score of 3.6. The segregation of symptom development of the Oh1VI x Va35 F2 seedlings fit 3:1 (p = 0.45), 13:3 (p =

0.70), and 11:5 (p = 0.08) susceptible: resistance ratios.

5.4.2. Selective mapping and phenotyping of RIL populations

One hundred and five RIL of each mapping population (Oh1VI x Oh28 and

Oh1VI x Va35) were selected based on genotypic data. In the case of the Oh1VI x Oh28 population, MapPop identified 89% of the selected lines in a single run. On average, the

138 selected lines had 25% more recombination events than the non-selected lines (data not shown). These informative progeny were used to conduct the gene mapping analysis.

Thirteen RIL selected from the Oh1VI x Oh28 population were omitted from the analysis because their seed mortality after VPI was higher than 40% in the three replications.

The distribution of means for disease incidence and AUDPC of the RILs inoculated with MNeSV ranged from completely resistant to extremely susceptible in both populations, and the distribution was binomial (Fig. 5.4). AUDPC values based on symptom severity correlated with disease incidence in both populations (p < 0.0001; r2 ≥

0.899) (data not shown). The percentage of the total variance explained for AUDPC and incidence due to genetic effects was 47 and 36%, respectively, for the Oh1VI x Oh28 population, and 34 and 32%, respectively for the Oh1VI x Va35 population. Variance due to a replication effect for both traits and populations was less than 16% (Table 5.2).

5.4.3. QTL mapping

A major QTL conferring resistance to MNeSV mapped to the long arm of chromosome 10 in both maize populations. In both populations, the resistant allele(s) were derived from Oh1VI. The QTL for reduced MNeSV incidence identified by CIM mapping in the Oh1VI x Oh28 population between markers PZA00647-9 and

PHM15868.56 explained 39% of the total phenotypic variance, and the QTL for reduced

MNeSV AUDPC explained 46% of the phenotypic variance (Table 5.3). These QTLs overlapped in a region covering 9.9 Mb of genomic DNA. The QTLs for reduced disease incidence and AUDPC identified by single marker regression in the Oh1VI x Va35

139 explained up to 74% and 70% of the total phenotypic variance, respectively (Table 5.4).

The SSR markers umc1930 and bnlg1215 located on maize bins 10.04 and 10.05, respectively had both the most significant linkage, explained the most variation, and were associated with the greatest reduction in disease incidence and AUDPC.

5.5. Discussion

MNeSV is an emerging virus disease of maize. Due to the severity of symptoms on susceptible sweet and dent corn, it has potential to cause severe yield losses (Louie et al., 2000; Redinbaugh and Pratt, 2009). In addition, MNeSV is related to viruses, including MCMV and MWLMV, that cause significant yield reductions in several regions around the world (Castillo and Hebert, 1974; Lapierre and Signoret, 2004;

Redinbaugh and Pratt, 2009; Uyemoto et al., 1980; Wangai et al., 2012). We have identified a major QTL explaining a large proportion of the resistance to MNeSV in two

RIL populations (Tables 5.3 and 5.4). While it is unknown if this QTL could confer resistance to the related MWLMV (genus Aureovirus; family Tombusviridae), a

Nebraska isolate of MCMV (genus ; family Tombusviridae) infects both

Oh1VI and Oh28 (unpublished results). There are several resistance loci in maize that confer or enhance resistance to multiple viruses in the same family. For example Wsm1, a locus on the short arm of chromosome 6, confers resistance to the Potyviridae Wheat streak mosaic virus (WSMV) (McMullen et al., 1994) and complete or partial resistance to other viruses in the same family, including: Maize dwarf mosaic virus (MDMV),

Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Johnsongrass

140 mosaic virus (JGMV) (Jones et al., 2011; Stewart et al., 2012). A major QTL on the long arm of chromosome 2 confers resistance to two viruses in the family Rhabdoviridae:

Maize mosaic virus (MMV) and Maize fine streak virus (MFSV) (Chapter 3). These results suggest that there could be a potential use of the mapped resistance locus for controlling both MNeSV and MWLMV.

MNeSV is a relatively new disease and only a few lines have been found to be resistant to the virus including Oh1VI and OSU23i (Louie et al., 2000; Redinbaugh and

Pratt, 2009). A major QTL explaining a large proportion of the total phenotypic variance was consistently mapped on the long arm of chromosome 10 using two populations derived from the resistant line Oh1VI (Table 5.3 and 5.4). The fact that we confirmed the location and effect of the QTL in two different susceptible genetic backgrounds suggests that this QTL could be effectively used to introgress resistance into diverse maize populations.

Interestingly, the position of the QTL conferring resistance to MNeSV was mapped near loci conferring resistance to diseases caused by phylogenetically distinct viruses (Fig. 5.5). Using the entire 256 Oh1VI x Oh28 RIL population, resistance loci for

WSMV, MDMV, and Maize rayado fino virus (MRFV) were mapped close or on the same genetic region than the MNeSV resistance QTL (Chapter 3). The location of the resistance loci for MDMV, WSMV and MRFV has been confirmed using diverse maize mapping populations (Jones et al., 2007; Jones et al., 2011; McMullen et al., 1994).

Additionally, mcd2, a locus conferring additive/recessive resistance to Maize chlorotic dwarf virus (MCDV) have also been mapped to the same region (Jones et al., 2004).

141

These results suggest that either a cluster of virus resistance genes located in this region of the maize genome (McMullen and Simcox, 1995; Redinbaugh et al., 2004; Wisser et al., 2006) or a single locus that is effective against multiple pathogens. Many R genes in plants are found in clusters that contain several copies of homologous genes arising from single or multiple gene families. The best characterized cluster of R genes in maize includes the maize Rp cluster mapped on bin 10.01. This region contains Rp1, Rp3, Rp4,

Rp5, and Rpp9, genes that confer resistance to either common rust (Puccinia sorghi) or southern rust (Puccinia polysora) (McMullen and Simcox, 1995; Smith et al., 2004).

Clustering of resistance genes has been explained by the presence of multi-gene families of resistance loci that expand due to mispairing during recombination followed by divergence over time (Friedman and Baker, 2007; Li et al., 2010; Michelmore and

Meyers, 1998).

Resistance to MNeSV derived from Oh1VI was partially expressed in the F1 progenies and segregated in the F2 generations (Table 5.1). Based on the results of the F1 seedlings, it is not clear if the resistance conferred by Oh1VI to MNeSV was recessive, additive, or dominant. At 23 dpi, 85% of the Oh1VI x Oh28 F1 seedlings were susceptible to the disease, suggesting that resistance was recessive, but symptom severity suggested resistance was additive, since F1 seedlings developed intermediate symptoms (severity rate = 3.2) relative to their parents (severity rate Oh1VI = 2.4, and Oh28 = 4.8). A different pattern was observed in the Oh1VI x Va35 screening. Disease incidence of the

Oh1VI x Va35 F1 seedlings suggested dominant resistance (F1 = 47%, Oh1VI = 48%,

Va35 = 100%), but severity ratings for symptomatic plants suggested recessive resistance 142

(F1 = 4.3, Oh1VI = 2.1, Va35 = 4.2). Differences between screening responses could be attributed to the use of a different susceptible genetic background and disease escapes associated with VPI (Jones et al., 2007; Jones et al., 2011; Louie, 1986; Louie, 1995).

Although it is not possible to identify the mode of action of the genes involved in the resistance to MNeSV, the severity scores of symptomatic F1 seedlings suggest that recessive or additive genetic effects are more likely to be responsible for the virus resistance observed in Oh1VI.

Although recessive and additive virus resistance are less common in maize, a similar genetic response was described for the resistance to MCDV in the F1 seedlings of

Oh1VI x Va35 and Oh1VI x Oh28 (Jones et al., 2004; Chapter 3). In these experiments, almost all of the F1 seedlings developed intermediate disease symptoms, compared with their parents, when inoculated with MCDV. Little is known about the molecular basis of

MCDV or any other virus disease resistance in maize. In most of characterized cases, recessive resistance to a virus corresponds to mutations in the eukaryotic translation factors, particularly those in the eIF4E gene family of translation initiation factors

(Gururani et al., 2012; Kang et al., 2005), but none of these factors have been mapped to this region of the maize genome according to the Maize Genetics and Genomics Database

(http://www.maizegdb.org). In other cases, proteins that interact with or are required for the replication or movements of viruses are host susceptibility factors. The Tom1 and

Tom2A genes in Arabidopsis thaliana encode a putative multipass transmembrane protein and a 280 amino acid putative four-pass transmembrane protein with a C-terminal farnesylation signal, respectively, that are required for Tobacco mosaic virus (TMV) to 143 replicate and cause disease. Mutations on these genes completely suppress replication of the virus. These mutated genes (tom1 and tom2A) interact with each other to enhance resistance. It is presumed that the transmembrane proteins encoded by Tom1 and Tom2A are essential constituents of the replication complex (Tsujimoto et al., 2003;

Yamanaka et al., 2002).

Mendelian segregation analysis of disease incidence in the Oh1VI x Oh28 F2 generation suggested that two genes were responsible for the susceptibility, one dominant and another unlinked recessive gene (dominant suppressor) (Russell, 2002). However, it is possible that disease escapes may have interfered with determining the ratio of resistant to susceptible plants. If we assumed a disease escape rate of 19% from the percentage of asymptomatic plants in the susceptible Oh28, the observed segregation would fit a 11:5 ratio (p > 0.9), suggesting that two dominant genes are required to confer resistance, but the combination of two recessive alleles cause intermediate disease symptoms (Weigel and Glazebrook, 2008). This model could also explain the response of the heterozygous

F1 seedlings to MNeSV. Assuming 15% escapes in the F1, most of the seedlings that showed disease symptoms had an intermediate response to MNeSV compared with the susceptible parent (Table 5.1). Segregation analysis of disease incidence in the Oh1VI x

Va35 F2 seedlings confirmed the 11:5 segregation model; however, a single gene model

(3:1 segregation) and two-gene model (13:3 segregation) were also possible. While it was not feasible to identify the mode of action of the genes involved in MNeSV resistance from this experiment, our results indicate that one or two-gene models are sufficient to explain the resistance conferred by Oh1V1. Additional analyses in other populations or 144 near isogenic lines are needed to ascertain the number of genes involved in MNeSV resistance and to clarify the mode of inheritance.

VPI facilitates mechanical transmission of several viral diseases in maize (Louie,

1995; Louie et al., 2000; Redinbaugh et al., 2001), but the relatively low efficiency of transmission of some viruses has prevented its use in genetic analysis. Although the rate of disease infection in the susceptible control (Oh28) was relatively low (59%, ranging from 30 to 100%, data not shown) when screening the RIL populations, the percentage of the total phenotypic variance explained by genetic effects was relatively high (>30%) allowing us to consistently identify a resistance locus in two independently evaluated populations. VPI has been used to inoculate viruses in other species such as barley

(Madriz-Ordenana et al., 2000), wheat, rice, and soybeans (Redinbaugh and Louie, unpublished results) and we demonstrated that it can be used for genetic analysis as well.

Two selective mapping strategies were used to identify the most informative individuals from larger mapping populations. The MapPop software (Brown and Vision, 2000) quickly and consistently found a reduced sample of lines known to have similar map resolution and QTL detection power than the larger population from which it was derived

(Brown et al., 2000; Vision et al., 2000; Xu et al., 2005). Zambrano (2012) and Simon et al. (2008) have demonstrated that the use of this approach does not impair the power of detection of QTL with large phenotypic effect.

The identification of a major loci conferring resistance to MNeSV in maize before the disease becomes economically important provides an opportunity to develop or identify resistant lines in advance. In addition, the identified resistance QTL could also

145 contribute to the control of another virus in the same genus, MWLMV that causes yield losses in sweet corn. Genetically, the identification of clusters of resistance genes to unrelated virus diseases in the maize genome provides additional evidence of the evolutionary patterns and distribution of resistance genes in maize.

5.6. Acknowledgments

I thank R. Louie (USDA-ARS, retired) for valuable discussion about the VPI technique. I thank Adriana Tomas (DuPont-Pioneer) and the Dupont Pioneer Marker Lab for genotyping the Oh1VI x Oh28 RIL population I also thank J. Abt, C. Nacci, K.

Morales, K. Willie and C. Wallace (USDA-ARS) for technical assistance. I also thank the

Instituto Nacional Autónomo de Investigaciones Agropecuarias (INIAP), Ecuador for a fellowship to support my Ph.D. study.

146

5.7. References

Balzarini, M., and Milligan, S. 2003. Best linear unbiased prediction (BLUP) for genotype performance. In: Kang M. S. (ed) Handbook of Formulas and Software for Plant Geneticists and Breeders. The Haworth Press, Inc, NY, pp 181-191.

Brown, D. G., and Vision, T. J. 2000. MapPop 1.0: Software for selective mapping and bin mapping. http://www.bio.unc.edu/faculty/vision/lab/mappop (accessed 11 April 2013).

Brown, D. G., Vision, T. J., and Tanksley, S. D. 2000. Selective mapping: a discrete optimization approach to selecting a population subset for use in a high-density genetic mapping project. 11th Annual ACM/SIAM Symposium on Discrete Algorithms. San Francisco, CA.

Castillo, J., and Hebert, T. T. 1974. A new virus disease of maize in Peru. Phytopathol. 9:79-84.

Changa, C. 1998. Susceptibility of resistant maize germplasm to maize streak geminivirus by vascular puncture inoculation. Dissertation. Columbus, OH: The Ohio State University.

Clewer, A. G., and Scarisbrick, D. H. 2001. Practical statistics and experimental design for plant and crop science. J. Wiley, Chichester; New York.

Edwards, M. D., Stuber, C. W., and Wendel, J. F. 1987. Molecular marker facilitated investigations of quantitative trait loci in maize .1. Numbers, genomic distribution and types of gene action. Genetics 116:113-125.

Friedman, A. R., and Baker, B. J. 2007. The evolution of resistance genes in multi- protein plant resistance systems. Curr. Opin. Genet. Dev. 17:493-499.

Gao Huijiang, and Yang Runqing. 2006. Composite interval mapping of QTL for dynamic traits. Chinese Science Bulletin 51:1857-1862.

Gururani, M. A., Venkatesh, J., Upadhyaya, C. P., Nookaraju, A., Pandey, S. K., and Park, S. W. 2012. Plant disease resistance genes: current status and future directions. Physiol. Mol. Plant Pathol. 78:51-65.

Howad, W., Yamamoto, T., Dirlewanger, E., Testolin, R., Cosson, P., Cipriani, G., Monforte, A. J., Georgi, L., Abbott, A. G., and Arus, P. 2005. Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map. Genetics 171:1305-1309. 147

Jones, M. W., Redinbaugh, M. G., Anderson, R. J., and Louie, R. 2004. Identification of quantitative trait loci controlling resistance to Maize chlorotic dwarf virus. Theor. Appl. Genet. 110:48-57.

Jones, M. W., Redinbaugh, M. G., and Louie, R. 2007. The Mdm1 locus and maize resistance to Maize dwarf mosaic virus. Plant Dis. 91:185-190.

Jones, M. W., Boyd, E. C., and Redinbaugh, M. G. 2011. Responses of maize (Zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the Potyviridae. Theor. Appl. Genet. 123:729-740.

Kang, B., Yeam, I., and Jahn, M. M. 2005. Genetics of plant virus resistance. Annu. Rev. Phytopathol. 43:581-621.

Lapierre, H., and Signoret, P. A., editors. 2004. Viruses and virus diseases of Poaceae (Gramineae). INRA Editions, Paris, France.

Li, J., Ding, J., Zhang, W., Zhang, Y., Tang, P., Chen, J., Tian, D., and Yang, S. 2010. Unique evolutionary pattern of numbers of gramineous NBS-LRR genes. Molecular Genetics and Genomics 283:427-438.

Louie, R. 1995. Vascular puncture of maize kernels for the mechanical transmission of Maize white line mosaic virus and other viruses of maize. Phytopathol. 85:139-143.

Louie, R. 1986. Effects of genotype and inoculation protocols on resistance evaluation of maize to Maize dwarf mosaic virus Strains. Phytopathol. 76:769-773.

Louie, R., Abt, J. J., Anderson R. J., Redinbaugh M. G., and Gordon D. T. 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family Tombusviridae. Plant Dis. 84:1133-1139.

Louie, R., Redinbaugh, M. G., Anderson, R. J., and Jones, M. W. 2002. Registration of maize germplasm Oh1VI. Crop Sci. 42:991-991.

Louie, R., and Abt, J. J. 2004. Mechanical transmission of Maize rough dwarf virus. Maydica 49:231-240.

Louie, R., Seifers, D. L., and Bradfute, O. E. 2006. Isolation, transmission and purification of the High Plains virus. J. Virol. Methods 135:214-222.

Madden,, L. V., Hughes G., and van den Bosch, F. 2007. The study of plant disease epidemics. American Phytopathological Society, St. Paul.

148

Madriz-Ordenana, K., Thordal-Christensen, H., Collinge, D. B., Ramirez, P., Rojas- Montero, R., and Lundsgaard, T. 2000. Mechanical transmission of Maize rayado fino marafivirus (MRFV) to maize and barley by means of the vascular puncture technique. Plant Pathol. 49:302-307.

McMullen,, M. D., Louie R., Simcox, K. D., and Jones, M. W. 1994. Three genetic loci control resistance to Wheat streak mosaic virus in the maize inbred Pa405. Mol. Plant- Microbe Interac. 7:708-712.

McMullen, M. D., and Simcox, K. D. 1995. Genomic organization of disease and insect resistance genes in maize. Mol. Plant-Microbe Interact. 8:811-815.

Michelmore, R. W., and Meyers, B. C. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res. 8:1113-1130.

Redinbaugh, M. G., Louie, R., Ngwira, P., Edema, R., Gordon, D., and Bisaro, D. 2001. Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation. J. Virol. Methods 98:135-143.

Redinbaugh, M. G., Houghton,, W., Salomon R., Creamer, R., Hogenhout, S. A., Gordon, D. T., Ackerman, J., Meulia, T., Seifers, D. L., Abt, J. J., Styer, W. E., and Anderson, R. J. 2002. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathol. 92:1167-1174.

Redinbaugh, M. G., Gingery, R. E., and Jones, M. W. 2004. The genetics of virus resistance in maize (Zea mays L.). Maydica 49:183-190.

Redinbaugh, M. G., and Pratt, R. C. 2009. Virus Resistance. In: Bennetzen J. L., andHake S. C., (eds) Handbook of maize: Its Biology. Springer, New York. pp 251-268

Russell P. 2002. iGenetics. Pearson Education, Inc., San Francisco, CA.

Simon, M., Loudet, O., Durand, S., Berard, A., Brunel, D., Sennesal, F., Durand-Tardif, M., Pelletier, G., and Camilleri, C. 2008. Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers. Genetics 178:2253-2264.

Smith, S. M., Pryor, A. J., and Hulbert, S. H. 2004. Allelic and haplotypic diversity at the Rp1 rust resistance locus of maize. Genetics 167:1939-1947.

Stewart, L. R., Md, A. H., Jones, M. W., and Redinbaugh, M. G. 2012. Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus. Mol. Breed. 31:289-297.

149

Tsujimoto, Y., Numaga, T., Ohshima, K., Yano, M., Ohsawa, R., Goto, D. B., Niato, S., and Ishikawa, M. 2003. Arabidopsis tobamovirus multiplication (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1. EMBO J. 22:335-343.

Uyemoto, J. K., Bockelman, D. L., and Claflin, L. E. 1980. Severe outbreak of corn lethal necrosis disease in Kansas. Plant Dis. 64:99-100. van Ooijen, J. W., Boer, M. P., Jansen, R. C., and Maliepaard, C. 2002. MapQTL 4.0, Software for the calculation of QTL positions on genetic maps. 4.0.

Vision, T. J., Brown, D. G., Shmoys, D. B., Durrett, R. T., and Tanksley, S. D. 2000. Selective mapping: a strategy for optimizing the construction of high-density linkage maps. Genetics 155:407-420.

Wangai, A. W., Redinbaugh, M. G., Kinyua, Z. M., Miano, D. W., Leley, P. K., Kasina, M., Mahuku, G., Scheets, K., and Jeffers, D. 2012. First report of Maize chlorotic mottle virus and Maize Lethal Necrosis in Kenya. Plant Dis. 96:1582-1582.

Weigel, D., and Glazebrook, J. 2008. Genetic analysis of Arabidopsis mutants. CSH protocols. doi: 10.1101/pdb.top35.

Weiland, J. J., and Edwards, M. C. 2011. Linear-motion tattoo machine and prefabricated needle sets for the delivery of plant viruses by vascular puncture inoculation. Eur. J. Plant Pathol. 131:553-558.

Wisser, R. J., Nelson, R. J., and Balint-Kurti P. 2006. The genetic architecture of disease resistance in maize: A synthesis of published studies. Phytopathol. 96:120-129.

Wu, J., Ding, J., Du, Y., Xu, Y., and Zhang, X. 2007. Genetic analysis and molecular mapping of two dominant complementary genes determining resistance to Sugarcane mosaic virus in maize. Euphytica 156:355-364.

Xu, Z. L., Zou, F., and Vision, T. J. 2005. Improving quantitative trait loci mapping resolution in experimental crosses by the use of genotypically selected samples. Genetics 170:401-408.

Yamanaka, T., Imai, T., Satoh, R., Kawashima, A., Takahashi, M., Tomita, K., Kubota, K., Meshi, T., Naito, S., and Ishikawa, M. 2002. Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes. J. Virol. 76:2491-2497.

Zambrano, J.L. 2012. Selective mapping tutorial . 2012. http://www.extension.org/pages/61700/selective-mapping-tutorial (accessed 11 April 2012). 150

Zambrano, J. L., Francis, M. D., and Redinbaugh, M. G. 2013. Identification of resistance to Maize rayado fino virus in maize inbred lines. Plant Dis. in press.

Zhang, F. Z., Wagstaff, C., Rae, A. M., Sihota, A. K., Keevil, C. W., Rothwell, S. D., Clarkson, G. J., Michelmore, R. W., Truco, M. J., Dixon, M. S., and Taylor, G. 2007. QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits. J. Exp. Bot. 58:1433-1449.

151

Genotype Na Survival Incidence Severity Severity classe c d b (%) mean (%) 1 2 3 4 5 Oh1VI x Oh28

F1 90 89 85 3.2 12 5 47 11 5 F2 195 91 84 3.4 28 23 71 25 30 Oh1VI 75 77 12 2.4 51 4 3 Oh28 75 21 81 4.8 3 1 12 Oh1VI x Va35

F1 60 97 47 4.3 31 1 4 8 14 F2 60 97 79 3.6 12 8 13 14 11 Oh1VI 60 70 48 2.1 22 18 2 Va35 60 70 100 4.2 12 22 23 aNumber of seeds inoculated. bPercentage of seed that germinated. cPercentage of seedlings showing any disease symptom / germinated seedlings. dMean severity of symptomatic seedlings evaluated at 23 and 21 dpi, for the Oh1VI x Oh28 and Oh1VI x Va35, respectively, using a 1 to 5 scale, where 1 = no disease symptoms and 5 = severe disease symptoms. eNumber of maize seedlings in each severity class at 23 or 21 dpi for Oh1VI x Oh28 and Oh1VI x Va35, respectively.

Table 5.1. Segregation of resistance to Maize necrotic streak virus in F1 and F2 progenies.

152

Oh1VI x Oh28a Oh1VI x Va35b Sources AUDPCc Incidenced AUDPC Incidence Genotype 173 310 42 336 Replication 20 133 11 7 Residual 174 413 69 699 aA RIL population of 92 lines. Fifteen seeds per line were inoculated in three independent replications. bA RIL population of 105 lines. Ten seeds per line were inoculated in three independent replications. cArea under disease curve was estimated from disease severity symptoms at 9, 16 and 23 days post inoculation. dDisease incidence evaluated at 23 dpi as the percentage of plants showing any disease symptom.

Table 5.2. Components of variance for area under the disease progress curve and disease incidence in two maize recombinant inbred line populations inoculated with Maize necrotic streak virus.

153

Bin Traita Most LODb Variance Flanking Physical position of likely Explained Markers QTL (Range)c position (%) (cM) 10.04 Incidence 52.6 7.9 39 PZA00647.9 - 127,5 Mb -137,5 Mb PHM15868.56 10.04 AUDPC 57.6 10.0 46 PHM13687.14 - 117,9 Mb -137,5 Mb PHM15868.56 aDisease incidence evaluated at 23 days post inoculation (dpi) as the percentage of plant showing disease symptoms. Area under disease progress curve (AUDPC) estimated from disease severity symptoms at 9, 16 and 23 dpi. bLogarithm of the odds (LOD), p < 0.001 based on permutation. cRange in pair of bases of the physical position of the QTLs estimated from the flanking markers on the B73 physical map from the Maize Genetic and Genomic Database (http://www.maizegdb.org/.).

Table 5.3. Location and genetic effects of a major quantitative trait locus associated with resistance to Maize necrotic streak virus identified by composite interval mapping in a maize recombinant inbred line population derived from a Oh1V1 x Oh28 cross.

154

Bin Marker Infection AUDPC F R2 c RRd SSe F R2 RR SS valueb value 10.04 umc1930 166 0.74 39 ± 12 80 ± 11 134 0.70 8 ± 3 22 ± 4 10.05 bnlg1250 166 0.74 39 ± 13 81 ± 11 134 0.70 8 ± 3 22 ± 4 10.06 umc2122 92 0.62 41 ± 15 82 ± 12 101 0.64 8 ± 5 22 ± 5 aDisease infection evaluated 23 days post inoculation (dpi) as the percentage of plant showing disease symptoms. Area under disease progress curve (AUDPC) estimated from disease severity symptoms at 9, 16 and 23 dpi. bF value from single marker regression analysis with a probability of F < 0.0001. cR-square value from single marker regression analysis. dRR, mean ± standard deviation of lines homozygous for the allele from the resistant parent. eSS, mean ± standard deviation of lines homozygous for the allele from the susceptible parent.

Table 5.4. Location and genetic effects of markers associated with resistance to Maize necrotic streak virus identified by single marker regression in a maize recombinant inbred line population derived from a Oh1VI x Va35 cross.

155

Fig. 5.1. Leaf of maize seedlings inoculated with Maize necrotic streak virus. In the 1 to 5 scale, 1 = no symptoms, 2 = mild symptoms, 3 = intermediate, 4 = moderately severe symptoms, and 5 = severe disease symptoms.

156

100

80

60

40

Disease Disease Incidence (%) 20

0 Oh1VI Oh28 F1 F2

9dpi 16dpi 23dpi

Fig. 5.2. Maize necrotic streak virus disease incidence in maize lines Oh1VI, Oh28, and their F1 and F2 generations. Disease incidence was evaluated at 9, 16, and 23 days post inoculation as the percentage of plants showing symptoms. Data presented is the mean + standard error of 5, 5, 6, and 13 replications for Oh1VI, Oh28, F1 and F2, respectively.

157

5.0

5) - 4.0

3.0

2.0 Disease Disease Severity (1

1.0 9dpi 16dpi 23dpi

Oh1VI Oh28 F1 F2

Fig. 5.3. Maize necrotic streak virus disease progress in maize lines Oh1VI, Oh28, and their F1 and F2 generations. Severity of symptoms were evaluated at 9, 16, and 23 days post inoculation (dpi) using a rating scale of disease severity from 1 to 5, where 1 = no disease symptoms and 5 = severe symptoms. Data presented is the mean ± standard error of plants that showed symptoms.

158

A

40 40

20 20

Frequency Frequency

0 0

0 40 100 0 15 30

Incidence AUDPC

B

40 40

20 20

Frequency Frequency

0 0

0 40 100 0 15 30

Incidence AUDPC

Fig. 5.4. Mean distribution of disease severity and area under the disease progress curve (AUDPC) at 23 days post inoculation evaluated on: A) 92 maize recombinant inbred lines (RIL) derived from Oh1VI x Oh28, and B) 105 RIL derived from Oh1VI x Va35 for reaction to Maize necrotic streak virus.

159

10

0.0 PZA02554.1

21.4 PHM2828-83 25.0 PHM5740-9 PZA01451-1 30.1 PHM15331-16 PZA02961-6 PHM3922-32 34.5 WSMV 36.5 PHM1812-32

36.7 PHM1155-14 MDMV 39.4 PZA00337-3

MRFV 43.3 PHM13687-14 MNeSV 47.6 PZA00647-9

55.7 umc1477 mcd2 61.0 PHM15868-56 65.2 bnlg1028

74.1 bnlg2190 80.6 PZA00130-9 85.0 PZA02969-9 85.4 PZA00007-1

107.1 PHM3736-11 113.4 PHM10750-26 115.8 PHM1506-23

Fig. 5.5. Location (cM) of a quantitative trait loci conferring resistance to Maize necrotic streak virus (MNeSV) mapped on chromosome 10 of the maize recombinant inbred line population Oh1VI x Oh28. Loci conferring resistance to Wheat streak mosaic virus (WSMV), Maize dwarf mosaic virus (MDMV), and Maize rayado fino virus (MRFV) previously mapped in the same population are also shown. The relative map position of mcd2, a locus conferring resistance to Maize chlorotic dwarf virus (MCDV), is shown to the right.

160

CHAPTER 6

AGRONOMIC EVALUATION OF SELECTIONS FROM A MAIZE

RECOMBINANT INBRED LINE POPULATION WITH MULTI-VIRUS

RESISTANCE IN ABSENCE OF VIRAL DISEASES

6.1. Abstract

Oh1VI is an inbred line of maize that is highly resistant to several virus diseases.

Unfortunately, it is derived from an exotic Caribbean population and is not adapted to temperate regions. A recombinant inbred line population derived from Oh1VI and the temperate virus susceptible line Oh28 was evaluated to identify improved recombinant lines with resistance to several virus diseases. Eight multi-virus resistant recombinant lines were selected based on both genotypic marker data and their responses to infection by Maize fine streak virus (MFSV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV), and Maize rayado fino virus

(MRFV). In absence of disease, none of the selected lines showed favorable agronomic traits. All the lines displayed low yield, late flowering time, and a long anthesis-silking interval compared with the adapted parent, Oh28. However, lines 61238, 61453, and

61399 had better agronomic characteristics than the exotic parent, Oh1VI. These

161 experiments provide agronomic information of multi-virus resistant lines that could be relevant for breeding purposes.

6.2. Introduction

The multi-virus resistant line Oh1VI is highly resistant to several phylogenetically distinct viruses, including viruses in the families Potyviridae, Secoviridae,

Rhabdoviridae, Tombusviridae, and Tymoviridae (Jones et al., 2004; Louie et al., 2000;

Redinbaugh et al., 2002; Zambrano et al., 2013). Multi-virus resistance in Oh1VI is conferred by a cluster of loci that mapped to regions of chromosomes 2, 3, 6, and 10.

Single nucleotide polymorphisms flanking resistance have been identified to facilitate breeding and selection (Chapters 3, 4, 5).

Oh1VI offers a potential resource for breeding programs seeking to protect vulnerable areas of the world from current or emerging viral diseases through genetic resistance. The genetic linkage of resistance loci provides an advantage to breeders since these loci could be introgressed rapidly into elite cultivars by marker assisted selection.

However, the tropical line Oh1VI is not adapted to temperate regions and linkage drag could discourage its use. In Wooster, Ohio, Oh1VI produces silks (female flowers) extremely late in the season (around 100 days after planting) and it is highly susceptible to ( maydis DC. Corda) infection (Louie et al., 2002). These and other agronomic deficiencies, such as poor grain filling, high grain moisture, high ear placement, long anthesis-silking interval, and poor root and stalk strength, are typical problems that breeders face when introducing tropical materials to temperate 162 environments (Castillo-Gonzalez and Goodman, 1989; Djemel et al., 2012; Tarter et al.,

2004). Therefore, crosses between tropical and temperate germplasm are often performed to gain disease resistance and adaptation to the target environment (Hallauer et al., 2010;

Tarter et al., 2004).

Recombinant inbred lines (RILs) derived from Oh1VI and the virus susceptible temperate line Oh28, their genotypic data, and their infection responses to virus inoculations are now available (Chapters 3, 4, 5). These resources can facilitate selection of segregating lines based on phenotypic and marker information. The objective of the research described in this chapter was to select recombinant lines with multiple virus resistance and superior agronomic traits that could be used for applied breeding purposes.

I hypothesized that among the 256 RILs, there will be individual lines that combine desirable virus resistance from Oh1VI with desirable agronomic traits derived from

Oh28. Field tests were conducted during 2011 and 2012 to evaluate the RILs and identify virus resistant lines with superior agronomic characteristics. These experiments provide agronomic information of multi-virus resistant lines that might be relevant for breeding purposes.

6.3. Material and methods

6.3.1. Plant material

A RIL maize population derived from the multi-virus resistant line Oh1VI (PI

614734) and the virus susceptible line Oh28 was generated as previously described

(Chapter 3). Briefly, the F1 cross was made in the summers of 1996 and 2003. Through

163

2006, 511 F2 ears were generated. Seeds of F2 plants were planted ear to row and successively self-pollinated every year. By 2010, 260 RILs were self-pollinated between seven to nine times without selection. The lines have been maintained by the Corn,

Soybean and Wheat Quality Research Unit (CSWQRU) at the OARDC.

6.3.2. Agronomic evaluation of the RIL population

Plant and ear heights, male and female flowering times, and smut incidence

(Ustilago maydis DC. Corda) were evaluated as described by CIMMYT (1995) in the entire RIL population using a randomized complete block design in the summers of 2011

(May 11th) and 2012 (May 11th) in Snyder Farm at OARDC, Wooster, OH. Year was considered the block effect. The parental lines Oh1VI and Oh28 were included as controls. Each experimental plot consisted of a 3.5 m-long row with 17 seeds of each line spaced at 0.20 m within row and 0.76 m between rows with a final plant density of

60,455 plants ha-1. Planting was done mechanically using a “Kinze” four row plot planter equipped with cones (Almaco, Ames, IA). The fields for both years were previously used for soybean testing. They were fertilized pre-planting with 225 kg ha-1 of 10-20-20 according to current management practices for maize inbred lines. The pre-emergent herbicides glyphosate (1.5 kg ha-1) and cloransulam-methyl (FirstRate, 0.5 kg ha-1) were applied around three weeks before planting, and Atrazine (2 l ha-1) and dimethenamid-P

(Establish ATZ, 3.5 l ha-1) were applied two days after planting. Manual weeding and irrigation with sprinklers were provided as needed. Plants within the test plots were self- pollinated to increase seed. Analysis of variance and Pearson correlations were conducted

164 for the six traits using the SAS procedures PROC GLM and PROC CORR, respectively

(version 9.2, SAS Institute Inc., Cary, NC). Comparisons of means were performed for each trait using Fisher’s least significance difference (LSD) test at P = 0.05 (Clewer and

Scarisbrick, 2001).

6.3.3. Selection of multi-virus resistant lines

A set of multi-virus resistant lines was selected from the entire RIL population based on their phenotypic responses to inoculation with Maize fine streak virus (MFSV),

Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV), and a preliminary response (data from one replication) to Maize rayado fino virus (MRFV). Two approaches were used to select the multi-virus resistant lines: 1) lines with less than 4% disease incidence for all the above-mentioned diseases were identified, then marker assisted selection was used to identify the lines carrying the multi-virus resistance alleles previously mapped on chromosomes 2, 3, 6, and 10 of the same RIL population (Zambrano, this thesis); 2) The “Fieldbook 5.1/7.1” software

(Banziger and Barreto, 1999) selection program was used to help identify the multi-virus resistant lines. A selection index was calculated with the program using the virus disease incidence data for the five viruses. In the program, the intensity of selection was kept the same for the five viruses. The phenotypic and genotypic information of the RILs were reported previously (Chapters 3, 4, 5).

165

6.3.4. Agronomic evaluation of the multi-virus resistance lines

A complete randomized block design with four replicates was used to evaluate grain yield, agronomic value, female flowering time, anthesis silking interval, and smut

(U. maydis) incidence of eight selected multi-virus resistant RILs. The progenitors

(Oh1VI and Oh28) and the maize inbred lines B73, B105, and Pa405 were included as controls to compare their agronomic performance with the selected lines. Grain yield was adjusted to 14% of seed moisture and agronomic value was evaluated using a 1 to 5 scale, where 1 = excellent and 5 = extremely poor (CIMMYT, 1995). Agronomic value represented an index of plant vigor, high of plant and flowering uniformity of the lines.

The experimental unit consisted of two 5 m-long rows of 26 plants each with same planting density indicated above. The plots were hand planted using a jab-type corn planter (ALMACO, Ames, IA) with 2 seeds per site. Two weeks after planting, the plots were thinned to 26 plants per row. The evaluation was conducted during the summer of

2012 (May 14th) at Snyder Farm, Wooster, OH. The data was first tested for normal distribution and homogeneity of variance using the PROC UNIVARIATE command in

SAS (version 9.2, SAS Institute Inc., Cary, NC). Then, an analysis of variance and Fisher

LSD mean comparison were performed for each trait.

166

6.4. Results

6.4.1. Agronomic evaluation of the RIL population

Analysis of variance showed significant differences (p < 0.001) among genotypes for all the measured traits. Differences were also observed for the year effect for all traits except for female flowering time, which was not different between years (p > 0.05) (data not shown). The mean flowering time of the population was consistent across years based in their similar means and ranges observed in 2011 and 2012 (Table 1). Plant height, ear height and smut incidence varied between years for the population. Means and ranges for plant and ear height were lower in 2011 than in 2012, and smut incidence mean and range were higher. The weather patterns for the two years were distinct, with 2011 being the wettest in recorded history for NE Ohio and 2012 being the hottest.

There were significant positive correlations among all the measured agronomic traits in the population (p < 0.05) (Table 2). Correlations were highest between male and female flowering time (r = 0.81) and plant and ear height (r = 0.74), and the lowest between male flowering time and ear height (r = 0.24).

6.4.2. Selection of multi-virus resistant lines

Fifteen recombinant inbred lines were resistant to MDMV, SCMV, MFSV,

WSMV and MRFV with a disease incidence < 4% (data not shown). From this group, marker assisted selection identified three lines (61236, 61453, and 61488) carrying the multi-virus resistance loci that were previously mapped on chromosome 2, 3, 6, and 10,

167 and two lines (61238 and 61465) that carried loci on chromosome 2, 6 and 10, but lacked the one on chromosome 3 (Chapters 3, 4 and 5).

Based on the disease incidence data for the RIL population, a multi-virus resistance index was calculated using the selection assistant program of the software

“Fieldbook” (Banziger and Barreto, 1999). The index ranged from 3.5 to 34.9 with the lowest value indicating multi-virus resistance and the highest value multi-virus susceptibility. The five lines with the lowest index (61238, 61323, 61399, 61482, and

61488) were selected. The lines 61238, and 61488 were identified by both selection methods. The index was not correlated with any of the agronomic traits that were measured in the population (p > 0.2), indicating that there was no correlation between resistance and the agronomic traits (Table 2).

Although all the selected lines were resistant to the virus diseases, they showed significant differences for male and female flowering times, plant and ear height, and smut incidence suggesting considerable genetic variation among them (Table 3). Only line 61236 did not differ from the adapted virus susceptible parent Oh28 for flowering time, plant and ear heights and smut incidence, indicating potential agronomic value.

6.4.3. Agronomic evaluation of the multi-virus resistance lines

This trial was conducted to measure grain yield of the selected multi-virus resistant lines, and to compare their agronomic performance with widely used inbred lines. The residual plots of the variance indicated a linear relationship between the observed and predicted residuals, and no clear pattern for the plots involving standardized

168 residuals. Thus, homogeneity of variance and normal distribution for all traits were assumed. Analysis of variance showed significant differences (p < 0.001) among the genotypes for all measured traits. Differences due to the replication effect were not significant (p > 0.05) for most of the variables, except for smut incidence and female flowering time (p = 0.02 for both). The control line B73 had the highest yield with 4.71 ton/ha followed by Oh28 with 3.43 ton/ha (Table 4). All the multi-virus resistance lines had significantly less yield than the virus susceptible parent Oh28 or the control line

B105. The yields of the multi-virus resistant lines ranged from 0.79 to 2.09 ton/ha. These yields were significantly higher than the 0.07 ton/ha obtained by their multi-virus resistance parent Oh1VI. Of the eight selected multi-virus resistant lines, only line 61236 showed no differences with B73 or Oh28 for agronomic value and smut incidence, but it showed significantly longer female flowering time and anthesis silking interval according to the mean separation with Fisher’s LSD.

6.5. Discussion

Oh1VI is highly resistant to several unrelated maize virus diseases (Jones et al.,

2004; Louie et al., 2000; Redinbaugh et al., 2002; Zambrano et al., 2013). The method that was used to develop this line could help to explain the presence of multi-virus resistance loci on its genome. Oh1VI was selected from a symptomless plant of a landrace population inoculated with MCDV. Then, this plant was self-fertilized and its progeny were tested for their responses to MCDV in several cycles of selection (Louie et al., 2002). MCDV resistance is quantitative in nature (conferred by several genes), and it

169 is possible that the selection pressure resulted in most or all of the MCDV resistance loci to be selected and fixed in this line. Quantitative trait loci conferring resistance to MCDV have been mapped to regions on chromosomes 2, 3, 4, 6, and 10 (Jones et al., 2004).

Interestingly, most of these regions are also known to contain resistance loci for other unrelated virus diseases in diverse germplasm (Dussle et al., 2000; Jones et al., 2004;

Jones et al., 2007; McMullen et al., 1994; Ming et al., 1997; Prazeres De Souza et al.,

2008; Redinbaugh and Pratt, 2009; Zhang et al., 2003). The tendency of resistance genes to cluster in certain regions of the plant genome (Michelmore and Meyers, 1998; Wisser et al., 2006) could have inadvertently facilitated the selection of additional resistance loci controlling unrelated viral diseases.

Unfortunately, the lack of adaptation observed in Oh1VI could discourage its use in temperate maize breeding programs. Flowering time reflects the adaptation of a maize plant to its environment (Buckler et al., 2009). Oh1VI develops silks (female flowers) very late in the season (Table 3 and 4) with a long anthesis-silking interval (Table 4), resulting in a very poor kernel set and, therefore, low yield (Table 4). Consequently, better temperate adapted multi-virus resistant lines are needed.

The RIL population derived from Oh1VI and Oh28 showed considerable phenotypic variation for all measured agronomic traits (Table 1), suggesting that the inherent genetic variation was suitable for selection. Additionally, no agronomic trait was correlated with the estimated virus resistance index (Table 2). Unfortunately, none of the selected multi-virus resistance lines was similar to the adapted parent, Oh28, in agronomic performance. However, line 61236 showed similar responses to Oh28 during

170 the evaluations of the entire RIL population in 2011 and 2012 (Table 3), but it had considerably lower yield and longer anthesis silking interval (Table 4). Poor agronomic performance is expected due to the high proportion of exotic alleles (≈50%) contributed from Oh1VI. Several studies have suggested that the proportion of exotic germplasm incorporated into adapted populations before performing selection should be lower than

25% (Albrecht and Dudley, 1987; Crossa and Gardner, 1987). Back crosses between the selected lines and the recurrent adapted parent Oh28 have been made in order to have a better-adapted foundation population with which to begin selection.

Despite their relatively poor agronomic performance, lines 61238, 61453, and

61399 had better agronomic characteristics than the exotic parent, Oh1VI (Table 3 and

4). Line 61238 was selected on the basis of both phenotypic performance and marker assisted selection. This line represents an alternative source of resistance for several viral diseases, since it is better adapted to temperate regions than its parent Oh1VI. Some drawbacks to using lines 61453 and 61399 that might prevent their utilization include: even though line 61453 that carries all the mapped multi-virus resistance alleles, it was not completely resistant to SCMV (disease incidence = 3.8%); and, line 61399 was selected using the multi-virus resistance index, but had the susceptible alleles (from

Oh28) on chromosomes 3, and 10.

The agronomic evaluation of a maize inbred line is only one measure of its performance. It is known that the value of a maize inbred line is not in its performance per se, but in its hybrid combinations (Shull, 1908). Additionally, virtually all maize in the temperate region is sold as F1 hybrids. Therefore it would be worth evaluating these

171 multi-virus resistant lines in a series of F1 hybrid tests with multiple heterotic testers to provide breeders information on effectively incorporating resistance into inbred line breeding programs. F1 hybrids between the selected lines and commercial lines representing diverse heterotic groups and whose patent expired are being made to accomplish this goal.

Nothing is known about the agronomic performance of Oh1VI or the selected multi-virus resistance lines in tropical or subtropical locations. Seed has been shipped to

Kenya and Ecuador and information about their adaptation and use in tropical breeding programs should be available soon.

6.6. Acknowledgments

I thank M. Jones (USDA-CSWQRU) for providing the seed of the RIL population and controls used in this study. Thanks to M. Jones, J. Todd, E. Brenner and K. Morales

(USDA-CSWQRU) for their help during planting, weeding, watering, evaluation, pollination and harvest of the RILs.

172

6.7. References

Albrecht, B., and J.W. Dudley. 1987. Evaluation for four maize populations containing different proportions of exotic germplasm. Crop Sci. 27:480-486.

Banziger, M., and H. Barreto. 1999. Manual de usuario para fieldbook 5.117.1 y alfa. Mexico, DF. : CIMMYT.

Buckler, E.S., J.B. Holland, P.J. Bradbury, C.B. Acharya, P.J. Brown, C. Browne, E. Ersoz, S. Flint-Garcia, A. Garcia, J.C. Glaubitz, M.M. Goodman, C. Harjes, K. Guill, D.E. Kroon, S. Larsson, N.K. Lepak, H. Li, S.E. Mitchell, G. Pressoir, J.A. Peiffer, M.O. Rosas, T.R. Rocheford, M. Cinta Romay, S. Romero, S. Salvo, H. Sanchez Villeda, H.S. da Silva, Q. Sun, F. Tian, N. Upadyayula, D. Ware, H. Yates, J. Yu, Z. Zhang, S. Kresovich, and M.D. McMullen. 2009. The genetic architecture of maize flowering time. Science 325:714-718.

Castillo-Gonzalez, F., and M.M. Goodman. 1989. Agronomic evaluation of Latin American maize accessions. Crop Sci. 29:853-861.

CIMMYT. 1995. Manejo de los ensayos e informe de los datos para el programa de ensayos internacionales de maiz del CIMMYT. Mexico, DF. : CIMMYT.

Clewer, A.G., and D.H. Scarisbrick. 2001. Practical statistics and experimental design for plant and crop science. J. Wiley, Chichester; New York.

Crossa, J., and C.O. Gardner. 1987. Introgression of an exotic germplasm for improving an adapted maize population. Crop Sci. 27:187-190.

Djemel, A., P. Revilla, L. Hanifi-Mekliche, R.A. Malvar, A. Alvarez, and L. Khelifi. 2012. Maize (Zea mays L.) from the saharan oasis: Adaptation to temperate areas and agronomic performance. Genet. Resour. Crop Evol. 59:1493-1504.

Dussle, C.M., A.E. Melchinger, L. Kuntze, A. Stork, and T. Luebberstedt. 2000. Molecular mapping and gene action of Scm1 and Scm2, two major QTL contributing to SCMV resistance in maize. Plant Breeding 119:299-303.

Hallauer, A.R., M.J. Carena, and J.B. Miranda Filho 2010. Quantitative genetics in maize breeding. Springer Science, LLC.

Jones, M.W., M.G. Redinbaugh, R.J. Anderson, and R. Louie. 2004. Identification of quantitative trait loci controlling resistance to Maize chlorotic dwarf virus. Theor. Appl. Genet. 110:48-57.

173

Jones, M.W., M.G. Redinbaugh, and R. Louie. 2007. The Mdm1 locus and maize resistance to Maize dwarf mosaic virus. Plant Dis. 91:185-190.

Louie, R., M.G. Redinbaugh, R.J. Anderson, and M.W. Jones. 2002. Registration of maize germplasm Oh1VI. Crop Sci. 42:991-991.

Louie, R., J.J. Abt, R.J. Anderson, M.G. Redinbaugh, and D.T. Gordon. 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family Tombusviridae. Plant Dis. 84:1133-1139.

McMullen, M.D., R. Louie, K.D. Simcox, and M.W. Jones. 1994. Three genetic loci control resistance to Wheat streak mosaic virus in the maize inbred Pa405. Molecular Plant-Microbe Interactions : MPMI 7:708-712.

Michelmore, R.W., and B.C. Meyers. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res. 8:1113-1130.

Ming, R., J.L. Brewbaker, R.C. Pratt, T.A. Musket, and M.D. McMullen. 1997. Molecular mapping of a major gene conferring resistance to Maize mosaic virus. Theor. Appl. Genet. 95:271-275.

Prazeres De Souza, I.R., A.R. Schuelter, C.T. Guimaraes, I. Schuster, E. De Oliveira, and M. G. Redinbaugh. 2008. Mapping QTL contributing to SCMV resistance in tropical maize. Hereditas (Lund) 145:167-173.

Redinbaugh, M.G., W. Houghton, R. Salomon, R. Creamer, S.A. Hogenhout, D.T. Gordon, J. Ackerman, T. Meulia, D.L. Seifers, J.J. Abt, W.E. Styer, and R.J. Anderson. 2002. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathol. 92:1167-1174.

Redinbaugh, M.G., and R.C. Pratt. 2009. Virus resistance. p. 251-268. In J.L. Bennetzen, and S.C. Hake (eds.) Handbook of maize: Its biology. Springer, New York.

Shull, G.H. 1908. The composition of a field of maize. Report American Breeders Association 4:296-301.

Tarter, J.A., M.M. Goodman, and J.B. Holland. 2004. Recovery of exotic alleles in semiexotic maize inbreds derived from crosses between Latin American accessions and a temperate line. Theor. Appl. Genet. 109:609-617.

Wisser, R.J., R.J. Nelson, and P. Balint-Kurti. 2006. The genetic architecture of disease resistance in maize: A synthesis of published studies. Phytopathol. 96:120-129.

174

Zambrano, J.L., M.D. Francis, and M.G. Redinbaugh. 2013. Identification of resistance to Maize rayado fino virus in maize inbred lines. Plant Dis. in press:.

Zhang, S.H., X.H. Li, Z.H. Wang, M.L. George, D. Jeffers, F.G. Wang, X.D. Liu, M.S. Li, and L.X. Yuan. 2003. QTL mapping for resistance to SCMV in Chinese maize germplasm . Maydica 48:307-312.

175

Statistic / Flowering time (days) Height (cm) Smut Year Male Female Plant Ear incidence (%)a Mean ± SDb 2011 85 ± 5 87 ± 5 159 ± 23 81 ± 18 15 ± 23 2012 83 ± 3 87± 4 180 ± 26 94 ± 23 9 ± 12 Range 2011 73 - 105 75 - 109 103 - 230 45 - 135 0 - 100 2012 74 - 91 76 - 103 93 - 270 50 - 198 0 – 71 Parental lines 2011 Oh1VI 103 ± 2 107 ± 2 166 ± 24 101 ± 17 68 ± 10 2012 Oh1VI 103 ± 1 110 ± 1 205 ± 17 56 ± 5 2011 Oh28 79 ± 2 81 ± 2 140 ± 10 61 ± 8 4 ± 6 2012 Oh28 80 ± 0 80 ± 1 160 ± 9 0 ± 0 aUstilago maydis incidence expressed as the percentage of plants showing galls on any part of the plant. bStandard deviation.

Table 6.1. Descriptive statistics for five agronomic traits measured in 256 maize recombinant inbred lines derived from Oh1VI and Oh28 evaluated during the summers of 2011 and 2012 at Snyder Farm (OARDC, Wooster, OH).

176

Male Female Smut Height Height ear flowering flowering incidence plant time time Female flowering 0.81171 time <.0001 Smut incidence 0.35178 0.26242 <.0001 <.0001 Height plant 0.10731 0.14200 0.12553 0.0145 0.0012 0.0041 Height ear 0.23662 0.20330 0.15813 0.73947 <.0001 <.0001 0.0003 <.0001 Resistance index -0.04401 0.03401 -0.00743 0.08541 -0.02591 0.5259 0.6241 0.9148 0.2 177 0.7089

Table 6.2. Pearson correlation coefficients and probabilities for multi-virus resistance index and five agronomic traits evaluated in 256 maize recombinant inbred lines during the summers of 2011 and 2012 at Snyder Farm (OARDC, Wooster, OH).

177

Genotype Selection Flowering time Height Smut methoda (days) (cm) incidence Male Female Plant Ear (%)b 61236 P-MAS 80 85 157 78 0 61238 P-MAS, SI 81 85 170 83 21 61323 SI 82 85 145 71 11 61399 SI 90 87 164 90 4 61453 P-MAS 90 94 167 80 0 61465 P-MAS 82 84 153 95 46 61482 SI 91 92 188 115 5 61488 P-MAS, SI 84 89 145 71 3 Oh1VI 103 107 166 101 68 Oh28 79 81 140 61 4 LSD (0.05)c 4 4 32 26 4 aMethod of selection for each line, P-MAS = phenotypic and marker assisted selection and SI = selection index based on phenotypic data as described in the Method section. bUstilago maydis incidence expressed as the number of plants showing galls on any part of the plant. cFisher’s least significance difference.

Table 6.3. Means for five agronomic traits of selected multi-virus resistant maize inbred lines derived from Oh1VI and Oh28 evaluated during the summers of 2011 and 2012 at Snyder Farm (OARDC, Wooster, OH).

178

Genotype Yield Agronomic Smut Female Anthesis (ton/ha)b value incidence flowering silking (1-5)c (%)d (days) interval (days) B73 4.71 1.3 0 81 1 Oh28 3.43 1.5 0 80 0 B105 2.86 1.6 0 84 4 61236 2.09 1.9 0 87 6 Pa405 2.08 1.9 2 72 2 61238 1.84 3.1 28 87 4 61323 1.80 2.4 32 88 7 61399 1.49 2.9 6 88 -1 61488 1.35 2.5 9 88 7 61465 1.34 3.5 53 87 4 61453 0.84 2.5 11 94 4 61482 0.79 3.6 24 95 6 Oh1VI 0.07 4.3 57 110 7 LSD (0.05)e 0.49 0.6 12 2 2 Genotype, Prob. F <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Replicate, Prob. F 0.12 0.24 0.02 0.02 0.32 aMeans of four replicates. bGrain yield adjusted to humidity of 14%. cAgronomic value of a maize line in a 1 to 5 scale, where 1 = excellent and 5 = extremely poor. dUstilago maydis incidence expressed as the number of plants showing galls on any part of the plant. eFisher least significance difference.

Table 6.4. Yield and other agronomic traits of 13 maize inbred lines evaluated during summer 2012 at Snyder Farm (OARDC, Wooster, OH)a.

179

CHAPTER 7

CONCLUSIONS

7.1. Conclusions

The genetic study of virus resistance in maize has traditionally focused on viruses of the family Potyviridae and a few other virus diseases. Prior to this study nothing was known about the genetics of resistance to Maize rayado fino virus (MRFV), Maize fine streak virus (MFSV), and Maize necrotic streak virus (MNeSV). The main objective of this research was to identify the loci or QTLs that confer resistance to eight diverse viruses, including MRFV, MFSV and MNeSV and some of the better studied virus diseases (Table 7.1), in the multiple virus-resistant maize inbred line Oh1VI. Further, I determined that previously identified cluster of resistance genes on specific regions of the maize genome contain resistance to newly tested viruses. This research followed the classical method for genetic analysis of disease resistance that begins with the identification of resistant and susceptible genotypes, the determination of the mode of inheritance of the resistance, and the mapping of resistance loci using recombinant populations, including but not limited to F2 and recombinant inbred line (RILs) populations.

180

Genetic resistance for most of the virus diseases in maize has been identified. In the case of MRFV, resistance had been identified in only a few genotypes. During this research project, novel sources of resistance to MRFV were identified in the inbred lines

Oh1VI, CML287, and Cuba. Based in the normal distribution of disease incidence and disease severity observed in the 36 evaluated maize accessions, resistance to MRFV in maize was quantitative. Resistance to MRFV found in the tropical inbred line Oh1VI was mainly dominant, since the vast majority of its derived F1 hybrids were resistant to the disease (Table 7.1). The segregation of the MRFV resistance in Oh1VI derived F2 plants were consistent with one or two resistance genes, suggesting that the resistance conferred by this line could be monogenic or bigenic.

The resistance observed in the Oh1VI derived F1 hybrids was dominant for the vast majority of viruses, including the three viruses in the Potyviridae {Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV) and Wheat streak mosaic virus

(WSMV)}, the two viruses in the Rhabdoviridae {Maize mosaic virus (MMV) and

MFSV}, and the virus in the Tymoviridae (MRFV) (Table 7.1). Recessive or additive virus resistance, depending on the trait or population was identified for the

Tombusviridae MNeSV and the Secoviridae Maize chlorotic dwarf virus (MCDV). These findings agree with current knowledge of virus resistance that indicates that most of the virus resistance in plants is dominant.

Mendelian segregation analysis conducted with F2 progenies derived from Oh1VI indicated that the resistance to the viruses was mainly monogenically or bigenically controlled. For most of the viruses I tested, one or two gene-models were sufficient to

181 explain the classical segregation of the resistance observed in the F2, except for the segregation of the resistance to WSMV, which was consistent with a three gene-model.

The multi-virus resistance observed in Oh1VI was controlled by one to three genes.

QTLs for reduced disease incidence and area under disease progress curve

(AUDPC) to the eight virus diseases were mapped on several regions of the maize genome using a RIL population derived from the tropical multi-virus resistant Oh1VI and the temperate virus susceptible Oh28 (Fig. 7.1). QTLs conferring resistance to the viruses in the Potyviridae (MDMV, SCMV, and WSMV) mapped to regions of chromosomes 3,

6, and 10, regions identified previously from diverse germplasm. The QTLs identified for

MCDV resistance on chromosome 2, 3, 6 confirmed the previously reported locations of

MCDV resistance QTLs in chromosomes 3 and 6. The location of a QTL conferring resistance to MMV on chromosome 3 was confirmed in the Oh1VI x Oh28 RIL population; however, additional QTLs were also found on chromosomes 1, 2, 3, and 6, in the same region where QTLs conferring resistance to other virus diseases have been mapped. QTLs conferring resistance to MFSV were found on chromosomes 2 and 6, and

QTLs conferring resistance to MRFV and MNeSV both mapped on the same region of chromosome 10. The location of the QTLs conferring resistance to MRFV and MNeSV were validated in populations derived from Ki11 x B73 and Oh1VI x Va35, respectively.

The mapping of the genes or QTLs conferring resistance to eight different viral diseases using the same population provided a unique opportunity to identify clusters of virus resistance genes from a single source. The presence of clusters of virus resistance

QTLs on chromosomes 3, 6, and 10 was confirmed. In addition, a novel cluster of QTLs

182 conferring resistance to rhabdoviruses was mapped on the long arm of chromosome 2.

The genetic architecture of the virus resistance observed in the RIL population agrees with the findings of cluster of resistance genes in diverse plant species and suggests a common mechanism involved in the origin and evolution of the resistance.

The Oh1VI x Oh28 RIL population is a valuable genetic resource that should be used in further studies of maize resistance. The availability of a genetic map and marker information on the population could facilitate the study of resistance to other diseases, including high plain virus, and rust that are also segregating in the population.

Soon, near isogenic lines for the QTL regions of chromosomes 2, 3, 6, and 10 will be available what will facilitate the narrowing and fine mapping of these regions. The particular characteristic of being resistance to several phylogenetically diverse viruses makes Oh1VI a perfect candidate for re-sequencing. Then, bioinformatics analysis comparing Oh1VI sequences with the reference maize genome B73 which is susceptible to most of the tested viruses should provide a better insight about the molecular basis behind resistance.

The identification of clusters of virus resistance genes or QTLs in the tropical maize inbred line Oh1VI could facilitate the introgression of multiple virus resistance into breeding populations; however, linkage drag might discourage its use in temperate regions. The agronomic characterization of inbred lines from the population that carry multi-virus resistance alleles and are more adapted to temperate growth conditions will be a valuable resource to enhance disease resistance in maize. Eight recombinant inbred lines that were resistant to multiple viruses or/and carried the Oh1VI virus resistance

183 alleles found on chromosomes 2, 3, 6, and 10 were identified and were proposed as alternative sources of resistance to multiple virus diseases. However, the agronomic performance of these lines remained poor when compared with the temperate adapted lines Oh28 and B73. Further back crosses carrying the virus resistance alleles in elite germplasm are needed to efficiently use the identified virus resistance QTLs in temperate maize breeding programs.

184

Acrona Virus Family Genus Inheritb

MDMV Maize dwarf mosaic virus Potyviridae Potyvirus Dominant

SCMV Sugarcane mosaic virus Potyviridae Potyvirus Dominant

WSMV Wheat streak mosaic virus Potyviridae Tritimovirus Dominant

MCDV Maize chlorotic dwarf virus Secoviridae Waikavirus Additive/recessive

MMV Maize mosaic virus Rhabdoviridae Nucleorhabdovirus Dominant

MFSV Maize fine streak virus Rhabdoviridae Nucleorhabdovirusc Dominant

MRFV Maize rayado fino virus Tymoviridae Marafivirus Dominant

MNeSV Maize necrotic streak virus Tombusviridae Tombusvirusc Additive/recessive aAcron. Virus acronym bMode of Inheritance of the resistance cPossible member, Unassigned genus

Table 7.1. Viruses of maize discussed in this dissertation and mode of inheritance of the resistance in the maize inbred line Oh1VI.

185

Fig. 7.1. Genetic distribution of QTLs conferring resistance to Maize chlorotic dwarf virus (MCDV), Maize mosaic virus (MMV), Maize fine streak virus (MFSV), Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV) and Wheat streak mosaic virus (WSMV), Maize rayado fino virus (MRFV), and Maize necrotic streak virus (MNesV) in the maize recombinant inbred line population Oh1VI x Oh28. Bars indicate LOD scores from composite interval mapping across the genetic map (cM) for each virus disease. Lines within the ring for each virus indicate a significance threshold for LOD scores of P < 0.01.

186

Bibliography

Agrios, George N. 2005. Plant Pathology Vol. 5th ed. Elsevier Academic Pres. Burlington, MA.

Albrecht, B., and J. W. Dudley. 1987. Evaluation for four maize populations containing different proportions of exotic germplasm. Crop Science 27: 480-6.

Ali, Farhan, and Jianbing Yan. 2012. Disease resistance in maize and the role of molecular breeding in defending against global threat. Journal of Integrative Plant Biology 54: 134-51.

Balint-Kurti, P., and G. S. Johal. 2009. Maize disease resistance. In Handbook of maize: Its biology., eds. J. L. Bennetzen, S. C. Hake. New York: Springer. pp. 229-250.

Balzarini, M., and S. Milligan. 2003. Best linear unbiased prediction (BLUP) for genotype performance. In Handbook of formulas and software for plant geneticists and breeders., ed. M. S. Kang, NY: The Haworth Press, Inc. pp. 181-191.

Banziger, M., and Barreto, H. Manual de usuario para fieldbook 5.117.1 y alfa.1999. CIMMYT, Mexico, DF.

Barreto, H., G. Edmeades, S. Chapman, and J. Crossa. 1997. The alpha lattice design in plant breeding and agronomy: Generation and analysis. In Developing drought and low N-tolerant maize., eds. G. Edmeades, M. Banziger, H. Mickelson and C. Peña-Valdivia,. Mexico DF: CIMMYT. pp. 544-551

Bendahmane, Abdelhafid, Konstantin Kanyuka, and David C. Baulcombe. 1999. The rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11: 781-91.

Bennetzen, J. L. 2009. Maize genome structure and evolution. In Handbook of maize: Genetics and genomics., eds. J. L. Bennetzen, S. C. Hake. New York, USA: Springer Science and Business Media LLC. pp. 179-199

187

Bockelman, D. L., L. E. Claflin, and J. K. Uyemoto. 1982. Host range and seed- transmission studies of Maize chlorotic mottle virus in grasses and corn. Plant Disease 66: 216-8.

Boller, Thomas, and Georg Felix. 2009. A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology 60: 379-406.

Bonamico, N. C., M. A. Di Renzo, M. A. Ibañez, M. L. Borghi, D. G. Díaz, J. C. Salerno, and M. G. Balzarini. 2012. QTL analysis of resistance to mal de río cuarto disease in maize using recombinant inbred lines. The Journal of Agricultural Science 150: 619-629.

Bonas, U., and Lahaye, T. 2002. Plant disease resistance triggered by pathogen-derived molecules: refined models of specific recognition. Curr Opin Microbiol 5:44-50.

Bradfute, O. E., R. W. Toler, C. W. Boothroyd, D. C. Robertson, L. R. Nault, and D. T. Gordon. 1979. Identification of Maize rayado fino virus in the United States. Plant Disease 64: 50-3.

Brewbaker, J. L. 1981. Resistance to Maize mosaic virus. In Virus and virus-like disease of maize in the united states., eds. D. T. Gordon, J. K. Knoke and G. E. Scott. 247th ed., Wooster, Ohio: Southern Cooperative Series Bulletin. pp. 145-151.

Brown, D. G., and T. J. Vision. 2000. MapPop 1.0: Software for selective mapping and bin mapping. Vol. 1.

Brown, D. G., T. J. Vision, and S. D. Tanksley. 2000. Selective mapping: A discrete optimization approach to selecting a population subset for use in a high-density genetic mapping project.

Buckler, Edward S., James B. Holland, Peter J. Bradbury, Charlotte B. Acharya, Patrick J. Brown, Chris Browne, Elhan Ersoz, et al. 2009. The genetic architecture of maize flowering time. Science 325: 714-8.

Bustamante, P. I., P. Ramirez, and R. Hammond. 1998. Evaluation of maize germ plasm for resistance to Maize rayado fino virus. Plant Disease 82: 50-6.

Carpane, Pablo D. 2007. Host resistance and diversity of Spiroplasma kunkelii as components of corn stunt disease. Dissertation, Oklahoma State University.

Castillo, J., and T. T. Hebert. 1974. A new virus disease of maize in Peru. Phytopathology 9: 79-84.

188

Caviedes, Mario. 1986. INIAP-180: Nueva variedad de maíz de alto rendimiento. INIAP, 180.

Chahal, G. S., and S. S. Gosal. 2002. Principles and procedures of plant breeding. United Kingdom: Alpha Science International Ltd.

Changa, C. 1998. Susceptibility of resistant maize germplasm to maize streak geminivirus by vascular puncture inoculation. Dissertation. The Ohio State University.

Chen, Yuting, Bryan J. Cassone, Xiaodong Bai, Margaret G. Redinbaugh, and Andrew P. Michel. 2012. Transcriptome of the plant virus vector graminella nigrifrons, and the molecular interactions of maize fine streak rhabdovirus transmission. Plos One 7: e40613.

Chicas, Mauricio, Mario Caviedes, Rosemarie Hammond, Kenneth Madriz, Federico Albertazzi, Heydi Villalobos, and Pilar Ramírez. 2007. Partial characterization of Maize rayado fino virus isolates from Ecuador: Phylogenetic analysis supports a central american origin of the virus. Virus Research 126: 268-76.

Chisholm, S. T., S. K. Mahajan, S. A. Whitham, M. L. Yamamoto, and J. C. Carrington. 2000. Cloning of the arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus. Proceedings of the National Academy of Sciences of the United States of America 97: 489-94.

Chisholm, S. T., M. A. Parra, R. J. Anderberg, and J. C. Carrington. 2001. Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus. Plant Physiology 127: 1667-75.

Chisholm, S. T., G. Coaker, B. Day, and B. J. Staskawicz. 2006. Host-microbe interactions: Shaping the evolution of the plant immune response. Cell 124: 803-14.

Churchill, G. A., and R. W. Doerge. 1994. Empirical threshold values for quantitative trait mapping. Genetics 138: 963-71.

CIMMYT. 1981. Report on maize improvement: 1980-81. Mexico, DF: CIMMYT.

CIMMYT. 1995. Manejo de los ensayos e informe de los datos para el programa de ensayos internacionales de maiz del CIMMYT. Mexico, DF.

Clewer, Alan G., and D. H. Scarisbrick. 2001. Practical statistics and experimental design for plant and crop science. Chichester; New York: J. Wiley.

189

Coaker, G. L. 2003. Genetic and biochemical characterization of resistance to bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. Dissertation. The Ohio State University.

Cosson, Patrick, Luc Sofer, Quang Hien Le, Valerie Leger, Valerie Schurdi-Levraud, Steven A. Whitham, Miki L. Yamamoto, et al. 2010. RTM3, which controls long- distance movement of potyviruses, is a member of a new plant gene family encoding a meprin and TRAF homology domain-containing protein. Plant Physiology 154: 222-32.

Cournoyer, Patrick, and Savithramma P. Dinesh-Kumar. 2011. NB-LRR immune receptors in plant virus defence, ed. Caranta, C Aranda, MA Tepfer, M LopezMoya,JJ.

Covey, S. N., N. S. AlKaff, A. Langara, and D. S. Turner. 1997. Plants combat infection by gene silencing. Nature 385: 781-2.

Crossa, J., and C. O. Gardner. 1987. Introgression of an exotic germplasm for improving an adapted maize population. Crop Science 27: 187-90.

Culver, James N., and Meenu S. Padmanabhan. 2007. Virus-induced disease: Altering host physiology one interaction at a time. Annual Review of Phytopathology 45: 221-43.

Dangl, J. L., and J. D. G. Jones. 2001. Plant pathogens and integrated defence responses to infection. Nature 411: 826-33.

De-Oliveira, Elizabeth, Aildson P. Duarte, Roberto De-Carvalho, and Antonio C. De- Oliveira. 2004. Molicutes e virus na cultura do milho no brasil: Caracterizacao e factores que afetam sua incidencia. In Doencas en milho. molicutes, virus, vetores e mancha por phaeosphaeria., eds. Elizabeth De-Oliveira, Charles M. De-Oliveira, 17. Brasilia: Embrapa Informacao Tecnologica.

Di Renzo, M. A., N. C. Bonamico, D. G. Diaz, M. A. Ibanez, M. E. Faricelli, M. G. Balzarini, and J. C. Salerno. 2004. Microsatellite markers linked to QTL for resistance to mal de rio cuarto disease in zea mays L. Journal of Agricultural Science 142: 289-95.

Diaz, J. A., C. Nieto, E. Moriones, and M. A. Aranda. 2002. Spanish melon necrotic spot virus isolate overcomes the resistance conferred by the recessive nsv gene of melon. Plant Disease 86: 694.

Diaz-Pendon, JA, V. Truniger, C. Nieto, J. Garcia-Mas, A. Bendahmane, and MA Aranda. 2004. Advances in understanding recessive resistance to plant viruses. Molecular Plant Pathology 5: 223-33.

190

Ding, Junqiang, Huimin Li, Yongxia Wang, Rongbing Zhao, Xuecai Zhang, Jiafa Chen, Zongliang Xia, and Jianyu Wu. 2012. Fine mapping of Rscmv2, a major gene for resistance to sugarcane mosaic virus in maize. Molecular Breeding 30: 1593-600.

Ding, Shou-Wei, and Olivier Voinnet. 2007. Antiviral immunity directed by small RNAs. Cell 130: 413-26.

Dintinger, J., D. Verger, S. Caiveau, A. M. Risterucci, J. Gilles, F. Chiroleu, B. Courtois, B. Reynaud, and P. Hamon. 2005. Genetic mapping of maize stripe disease resistance from the mascarene source. Theoretical and Applied Genetics 111: 347-59.

Djemel, A., P. Revilla, L. Hanifi-Mekliche, R. A. Malvar, A. Alvarez, and L. Khelifi. 2012. Maize (zea mays L.) from the saharan oasis: Adaptation to temperate areas and agronomic performance. Genetic Resources and Crop Evolution 59: 1493-504.

Dussle, C. M., A. E. Melchinger, L. Kuntze, A. Stork, and T. Luebberstedt. 2000. Molecular mapping and gene action of Scm1 and Scm2, two major QTL contributing to SCMV resistance in maize. Plant Breeding 119: 299-303.

Edwards, M. D., C. W. Stuber, and J. F. Wendel. 1987. Molecular-marker-facilitated investigations of quantitative-trait loci in maize .1. numbers, genomic distribution and types of gene-action. Genetics 116: 113-25.

Edwards, M. L., and J. I. Cooper. 1985. Plant virus detection using a new form of indirect ELISA. Journal of Virological Methods 11: 309-19.

Edwards, Michael C., and John J. Weiland. 2011. Presence of a polyA tail at the 3' end of maize rayado fino virus RNA. Archives of Virology 156: 331-4.

Espinoza, A. M., and R. Gamez. 1980. La ultraestructura de la superficie foliar de cultivares de maiz infectados con el virus del rayado fino. Turrialba 30: 413-20.

Farnham, D. E., G. O. Benson, and R. B. Pearce. 2003. Corn perspective and culture. In Corn: Chemistry and technology., eds. Pamela J. White, A. Johnson Lawrence. Second ed., 1. Min.: St. Paul. American Association of Cereal Chemists.

Flor, H. H. 1971. Current status of the gene for gene concept. Annu Rev of Phytopathology 9: 275-296.

Forrest Troyer, A. 2004. Persistent and popular germplasm in seventy centuries of corn evolution. In Corn :Origin, history, technology, and production., eds. C. Wayne Smith, Javier Betran and E. C. A. Runge, 133. Hoboken, N.J.: Wiley.

191

Friedman, Aaron R., and Barbara J. Baker. 2007. The evolution of resistance genes in multi-protein plant resistance systems. Current Opinion in Genetics & Development 17: 493-9.

Gamez, R. 1983. The ecology of Maize rayado fino virus in the American tropics. In Plant virus epidemiology., eds. R. T. Plumb, J. M. Thresh, 268-274. Oxford: Blackwell Scientific Publications.

Gamez, R., and F. Saavedra. 1986. Maize rayado fino: A model of a leafhopper-borne virus disease in the neotropics. In Plant virus epidemics : Monitoring, modelling and predicting outbreaks / edited by george D. McLean, ronald G. garrett, william G. ruesink., 315-326Sydney : Academic Press, c1986.

Gamez, R., and P. Leon. 1988. Maize rayado fino and related viruses. In Polyhedral virions with monopartite RNA genomes / edited by renate koenig., 213-233New York : Plenum Press, c1988.

Gao Huijiang, and Yang Runqing. 2006. Composite interval mapping of QTL for dynamic traits. Chinese Science Bulletin 51: 1857-62.

Gao, Zhihuan, Elisabeth Johansen, Samantha Eyers, Carole L. Thomas, T. H. Noel Ellis, and Andrew J. Maule. 2004. The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor elF4E in cell-to-cell trafficking. Plant Journal 40: 376-85.

Gebhardt, Christiane, and Jari P. T. Valkonen. 2001. Organization of genes controlling disease resistance in the potato genome. Annual Review of Phytopathology 39: 79-102.

Gimenez-Pecci, M., C. F. Nome, I. G. Laguna, C. Borgogno, E. Oliveira, and R. Resende. 2000. Occurrence of maize rayado fino virus in maize in argentina. Plant Disease 84: 1046-.

Gomez, P., A. M. Rodriguez-Hernandez, B. Moury, and M. A. Aranda. 2009. Genetic resistance for the sustainable control of plant virus diseases: Breeding, mechanisms and durability. European Journal of Plant Pathology 125: 1-22.

Gordon, D. T., and R. E. Gingery. 1977. Purification and chemical and physical properties of a U. S. isolate of maize rayado fino virus. Proceedings of the American Phytopathological Society: 171.

Gordon, D. T., O. E. Bradfute, R. E. Gingery, J. K. Knoke, L. R. Nault, and G. E. Scott. 1981. Introduction: History, geographical distribution, pathogen characteristics and economic importance. In Virus and virus-like disease of maize in the united states., eds.

192

D. T. Gordon, J. K. Knoke and G. E. Scott. 247th ed., 1-12. Wooster, Ohio: Southern Cooperative Series Bulletin.

Gordon, D. T., and G. Thottappilly. 2003. Maize and sorghum. In Virus and virus-like diseases of major crops in developing countries., eds. G. Loebenstein, G. Thottappilly, 295-334. The Netherlands: Kluwer Academic Publishers.

Grau, C. R., V. L. Radke, and F. L. Gillespie. 1982. Resistance of soybean cultivars to sclerotinia sclerotiorum. Plant Disease 66 : 506-8.

Guest, D., and J. F. Brown. 1997. Plant defenses against pathogens. In Plant pathogens and plant diseases., eds. J. F. Brown, H. J. Ogle, 263-286. Australia: Rockvale Publications.

Gururani, Mayank Anand, Jelli Venkatesh, Chandrama Prakash Upadhyaya, Akula Nookaraju, Shashank Kumar Pandey, and Se Won Park. 2012. Plant disease resistance genes: Current status and future directions. Physiological and Molecular Plant Pathology 78:51-65.

Hallauer, Arnel R., Marcelo J. Carena, and J. B. Miranda Filho, eds. 2010. Quantitative genetics in maize breeding. Handbook of Plant Breeding., eds. Jaime Prohens, Fernando Nuez and Marcelo J. Carena. Vol. 6. LLC: Springer Science.

Hamilton, John P., and C. Robin Buell. 2012. Advances in plant genome sequencing. Plant Journal 70: 177-90.

Hammond, R. W., and I. P. Bedendo. 2001. Role of maize rayado fino virus in the etiology of "red stunt" disease in brazil. Plant Disease 85: 99.

Hammond, RW, and P. Ramirez. 2001. Molecular characterization of the genome of maize rayado fino virus, the type member of the genus marafivirus. Virology 282: 338- 47.

Howad, W., T. Yamamoto, E. Dirlewanger, R. Testolin, P. Cosson, G. Cipriani, A. J. Monforte, L. Georgi, A. G. Abbott, and P. Arus. 2005. Mapping with a few plants: Using selective mapping for microsatellite saturation of the prunus reference map. Genetics 171: 1305-9.

Hulbert, S., Webb, C., Smith, S., and Sun, Q. 2001. Resistance gene complexes: Evolution and utilization. Annu Rev Phytopathol. 39:285-312.

Hull, R. 2002. Matthew's plant virology. San Diego: Academic Press.

193

Hunt, R. E., L. R. Nault, and R. E. Gingery. 1988. Evidence for infectivity of maize chlorotic dwarf virus and for a helper component in its leafhopper transmission. Phytopathology 78: 499-504.

Ingvardsen, Christina Roenn, Yongzhong Xing, Ursula Karoline Frei, and Thomas Luebberstedt. 2010. Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize. Theoretical and Applied Genetics 120: 1621-34.

Ioannidou, D., A. Pinel, C. Brugidou, L. Albar, N. Ahmadi, A. Ghesquiere, M. Nicole, and D. Fargette. 2003. Characterisation of the effects of a major QTL of the partial resistance to rice yellow mottle virus using a near-isogenic-line approach. Physiological and Molecular Plant Pathology 63: 213-21.

Ishibashi, Kazuhiro, Kiyoshi Masuda, Satoshi Naito, Tetsuo Meshi, and Masayuki Ishikawa. 2007. An inhibitor of viral RNA replication is encoded by a plant resistance gene. Proceedings of the National Academy of Sciences of the United States of America 104: 13833-8.

Jansen, R. C., and P. Stam. 1994. High-resolution of quantitative traits into multiple loci via interval mapping. Genetics 136: 1447-55.

Jeffers, Daniel P. 2004. Disease control. In Corn :Origin, history, technology, and production., eds. C. Wayne Smith, Javier Betrán and E. C. A. Runge, 669. Hoboken, N.J.: Wiley.

Johal, Durmukh S., and Steven P. Briggs. 1992. Reductase activity encoded by the HM1 disease resistance gene in maize. Science (Washington D C) 258: 985-7.

Jones, E. S., H. Sullivan, D. Bhattramakki, and J. S. C. Smith. 2007. A comparison of simple sequence repeat and single nucleotide polymorphism marker technologies for the genotypic analysis of maize (zea mays L.). Theoretical and Applied Genetics 115: 361- 71.

Jones, Elizabeth, Wen-Chy Chu, Mulu Ayele, Julie Ho, Ed Bruggeman, Ken Yourstone, Antoni Rafalski, et al. 2009. Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (zea mays L.) germplasm. Molecular Breeding 24:165-76.

Jones, J.D.G., and Dangl, J.L. 2006. The plant immune system. Nature 444:323-329.

Jones, M. W., M. G. Redinbaugh, R. J. Anderson, and R. Louie. 2004. Identification of quantitative trait loci controlling resistance to maize chlorotic dwarf virus. TAG.Theoretical and Applied Genetics.Theoretische Und Angewandte Genetik 110: 48- 57. 194

Jones, M. W., M. G. Redinbaugh, and R. Louie. 2007. The Mdm1 locus and maize resistance to maize dwarf mosaic virus. Plant Disease 91: 185-90.

Jones, M. W., E. C. Boyd, and M. G. Redinbaugh. 2011. Responses of maize (zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the potyviridae. Phytopathology 123: 729-740.

Kang, Byoung-Cheorl, Inhwa Yeam, and Molly M. Jahn. 2005. Genetics of plant virus resistance. Annual Review of Phytopathology 43 : 581-621.

Kang, L., J. X. Li, T. H. Zhao, F. M. Xiao, X. Y. Tang, R. Thilmony, S. Y. He, and J. M. Zhou. 2003. Interplay of the arabidopsis nonhost resistance gene NHO1 with bacterial virulence. Proceedings of the National Academy of Sciences of the United States of America 100: 3519-24.

Kao, C. H., and Z. B. Zeng. 2002. Modeling epistasis of quantitative trait loci using cockerham's model. Genetics 160: 1243-61.

Kogel, R., P. Ramirez, and R. W. Hammond. 1996. Incidence and geographic distribution of maize rayado fino virus (MRFV) in latin america. Plant Disease 80: 679-83.

Kosambi, D. D. 1944. The estimation of map distances from recombination values. Ann Eugenics 12 : 172-175.

Krzywinski, Martin I., Jacqueline E. Schein, Inanc Birol, Joseph Connors, Randy Gascoyne, Doug Horsman, Steven J. Jones, and Marco A. Marra. 2009. Circos: An information aesthetic for comparative genomics. Genome Research (June 18).

Kump, K.L., Bradbury, P.J., Wisser, R.J., Buckler, E.S., Belcher, A.R., Oropeza-Rosas, M.A., Zwonitzer, J.C., Kresovich, S., McMullen, M.D., Ware, D., Balint-Kurti, P.J., and Holland, J.B. 2011. Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population. Nat Genet 43:161-169.

Kyetere, D. T., R. Ming, M. D. McMullen, R. C. Pratt, J. Brewbaker, and T. Musket. 1999. Genetic analysis of tolerance to maize streak virus in maize. Genome 42: 20-6.

Lanfermeijer, F. C., J. Dijkhuis, M. J. G. Sturre, P. de Haan, and J. Hille. 2003. Cloning and characterization of the durable tomato mosaic virus resistance gene tm-2(2) from lycopersicon esculentum. Plant Molecular Biology 52: 1037-49.

Lanferrneijer, Frank C., Guoyong Jiang, Margriet A. Ferwerda, Jos Dijkhuis, Peter de Haan, Rencui Yang, and Jacques Hille. 2004. The durable resistance gene tm-22 from tomato confers resistance against ToMV in tobacco and preserves its viral specificity. Plant Science (Oxford) 167: 687-92. 195

Lapierre, H., and P. A. Signoret, eds. 2004. Viruses and virus diseases of poaceae (gramineae). Paris: INRA Editions.

Le Gall, Olivier, Miguel A. Aranda, and Carole Caranta. 2011. Plant resistance to viruses mediated by translation initiation factors, ed. Caranta, C Aranda, MA Tepfer, M LopezMoya,JJ.

Leon, P., and R. Gamez. 1981. Some physicochemical properties of maize rayado fino virus. Journal of General Virology 16: 67-75.

Li, Jing, Jing Ding, Wen Zhang, Yuanli Zhang, Ping Tang, Jian-Qun Chen, Dacheng Tian, and Sihai Yang. 2010. Unique evolutionary pattern of numbers of gramineous NBS-LRR genes. Molecular Genetics and Genomics 283: 427-38.

Loesch, P. J., and M. S. Zuber. 1967. An inheritance study of resistance to maize dwarf mosaic virus in corn (zea mays L). Agronomy Journal 59: 423.

Louie, R., and J. K. Knoke. 1981. Symptoms and disease diagnostic. In Virus and virus- like disease of maize in the united states., eds. D. T. Gordon, J. K. Knoke and G. E. Scott. 247th ed., 13-18. Wooster, Ohio: Southern Cooperative Series Bulletin.

Louie, R., J. K. Knoke, and D. L. Reichard. 1983. Transmission of maize-dwarf mosaic- virus with solid-stream inoculum. Plant Disease 67: 1328-31.

Louie . 1986. Effects of genotype and inoculation protocols on resistance evaluation of maize to maize-dwarf mosaic-virus strains. Phytopathology 76: 769-73.

Louie, R., and R. J. Anderson. 1993. Evaluation of maize chlorotic dwarf virus resistance in maize with multiple inoculations by graminella nigrifrons (homoptera: Cicadellidae). Journal of Economic Entomology: 1579-83.

Louie, R. 1995. Vascular puncture of maize kernels for the mechanical transmission of maize white line mosaic-virus and other viruses of maize. Phytopathology 85: 139-43.

Louie, R., J. J. Abt, R. J. Anderson, M. G. Redinbaugh, and D. T. Gordon. 2000. Maize necrotic streak virus, a new maize virus with similarity to species of the family tombusviridae. Plant Disease: 1133-9.

Louie, R., M. G. Redinbaugh, R. J. Anderson, and M. W. Jones. 2002. Registration of maize germplasm Oh1VI. Crop Science 42: 991.

Louie, R., and J. J. Abt. 2004. Mechanical transmission of maize rough dwarf virus. Maydica 49: 231-40.

196

Louie, Raymond, Dallas L. Seifers, and Oscar E. Bradfute. 2006. Isolation, transmission and purification of the high plains virus. Journal of Virological Methods 135: 214-22.

Lozano, Roberto, Olga Ponce, Manuel Ramirez, Nelly Mostajo, and Gisella Orjeda. 2012. Genome-wide identification and mapping of NBS-encoding resistance genes in solanum tuberosum group phureja. Plos One 7: e34775.

Lu Xiang-Ling, Li Xin-Hai, Xie Chuan-Xiao, Hao Zhuan-Fang, Ji Hai-Lian, Shi Li-Yu, and Zhang Shi-Huang. 2008. Comparative QTL mapping of resistance to sugarcane mosaic virus in maize based on bioinformatics. Yichuan 30: 101-8.

Madden, Laurence V., Gareth Hughes, and Frank van den Bosch. 2007. The study of plant disease epidemics. St. Paul: American Phytopathological Society.

Madriz-Ordenana, K., H. Thordal-Christensen, D. B. Collinge, P. Ramirez, R. Rojas- Montero, and T. Lundsgaard. 2000. Mechanical transmission of maize rayado fino marafivirus (MRFV) to maize and barley by means of the vascular puncture technique. Plant Pathology 49: 302-7.

Mansfield, J.W. 2009. From bacterial avirulence genes to effector functions via the hrp delivery system: an overview of 25 years of progress in our understanding of plant innate immunity. Molecular Plant Pathology 10:721-734.

Marcon, A., S. M. Kaeppler, S. G. Jensen, L. Senior, and C. Stuber. 1999. Loci controlling resistance to high plains virus and wheat streak mosaic virus in a B73 x Mo17 population of maize. Crop Science 39: 1171-7.

Martin, T., J. A. Franchino, E. D. Kreff, A. M. Procopiuk, A. Tomas, S. D. Luck, and G. G. Shu. 2009. Major QTL conferring resistance of corn to fijivirus. US Patent filed 2009.

McDowell, J. M., and B. J. Woffenden. 2003. Plant disease resistance genes: Recent insights and potential applications. Trends in Biotechnology 21: 178-83.

McMullen, M. D., and R. Louie. 1991. Identification of a gene for resistance to wheat streak mosaic virus in maize. Phytopathology 81: 624-7.

McMullen, M. D., R. Louie, K. D. Simcox, and M. W. Jones. 1994. Three genetic loci control resistance to wheat streak mosaic virus in the maize inbred Pa405. Molecular Plant-Microbe Interactions : MPMI 7: 708-12.

McMullen, M. D., and K. D. Simcox. 1995. Genomic organization of disease and insect resistance genes in maize. Molecular Plant-Microbe Interactions 8: 811-5.

197

McMullen, M. D., B. A. Roth, and R. Townsend. 1996. Maize chlorotic dwarf virus and resistance thereto. US Patent 5, filed 1996, and issued Oct. 29, 1996.

McMullen, Michael D., Stephen Kresovich, Hector Sanchez Villeda, Peter Bradbury, Huihui Li, Qi Sun, Sherry Flint-Garcia, et al. 2009. Genetic properties of the maize nested association mapping population. Science 325: 737-40.

Melchinger, A. E., L. Kuntze, R. K. Gumber, T. Lubberstedt, and E. Fuchs. 1998. Genetic basis of resistance to sugarcane mosaic virus in european maize germplasm. Theoretical and Applied Genetics 96: 1151-61.

Michelmore, R. 1995. Molecular approaches to manipulation of disease resistance genes. Annual Review of Phytopathology 33: 393-427.

Michelmore, R. W., and B. C. Meyers. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Research 8: 1113-30.

Ming, R., J. L. Brewbaker, R. C. Pratt, T. A. Musket, and M. D. McMullen. 1997. Molecular mapping of a major gene conferring resistance to maize mosaic virus. Theoretical and Applied Genetics 95: 271-5.

Mysore, K. S., and C. M. Ryu. 2004. Nonhost resistance: How much do we know? Trends in Plant Science 9: 97-104.

Nault, L. R., and O. E. Bradfute. 1979. Corn stunt: Involvement of a complex of leafhopper-borne pathogens. In Leafhopper vectors and plant disease agents., eds. K. Maramorosch, K. F. Harris, 561-586. New York: Academic Press.

Nault, L. R., D. T. Gordon, and R. E. Gingery. 1980. Leafhopper transmission and host range of maize rayado fino virus. Phytopathology 70: 709-12.

Nault, L. R., and J. K. Knoke. 1981. Maize vectors. In Virus and virus-like diseases of maize in the united states., eds. J. K. Knoke, D. T. Gordon and G. E. Scott. Vol. 247, 77- 84. Wooster, Ohio: South. Coop. Ser. Bull.

Nei, M., and Rooney, A. 2005. Concerted and birth-and-death evolution of multigene families. Annu Rev Genet 39:121-152.

Ng, James C. K., and Bryce W. Falk. 2006. Virus-vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annual Review of Phytopathology 44: 183-212.

198

Nieto, Cristina, Monica Morales, Gisella Orjeda, Christian Clepet, Amparo Monfort, Benedicte Sturbois, Pere Puigdomenech, et al. 2006. An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. Plant Journal 48: 452-62.

Palaniswamy, Usha R., and Kodiveri Muniyappa Palaniswamy. 2006. Handbook of statistics for teaching and research in plant and crop science. New York: Food Products Press.

Patterson, H. D., and E. R. Williams. 1976. A new class of resolvable incomplete block designs. Biometrika 63: 83-92.

Pernet, A., D. Hoisington, J. Dintinger, D. Jewell, C. Jiang, M. Khairallah, P. Letourmy, J. L. Marchand, J. C. Glaszmann, and D. G. de Leon. 1999. Genetic mapping of maize streak virus resistance from the mascarene source. II. resistance in line CIRAD390 and stability across germplasm. Theoretical and Applied Genetics 99: 540-53.

Pernet, A., D. Hoisington, J. Franco, M. Isnard, D. Jewell, C. Jiang, J. L. Marchand, B. Reynaud, J. C. Glaszmann, and D. G. de Leon. 1999. Genetic mapping of maize streak virus resistance from the mascarene source. I. resistance in line D211 and stability against different virus clones. Theoretical and Applied Genetics 99: 524-39.

Pokorny, R., and M. Porubova. 2006. Heritability of resistance in maize to the czech isolate of sugarcane mosaic virus. Cereal Research Communications 34: 1081-6.

Poland, Jesse A., Richard C. Pratt, Rebecca J. Nelson, Peter Balint-Kurti, and Randall J. Wisser. 2009. Shades of gray: The world of quantitative disease resistance. Trends in Plant Science 14: 21-9.

Poland, J.A., Bradbury, P.J., Buckler, E.S., and Nelson, R.J. 2011. Genome-wide nested association mapping of quantitative resistance to northern leaf blight in maize. Proc Natl Acad Sci. 108:6893-6898.

Prazeres De Souza, Isabel Regina, Adilson Ricken Schuelter, Claudia Teixeira Guimaraes, Ivan Schuster, Elizabeth De Oliveira, and Margaret Redinbaugh. 2008. Mapping QTL contributing to SCMV resistance in tropical maize. Hereditas (Lund) 145: 167-73.

Quint, M. 2003. Resistance gene analogs as a tool for basic and applyed resistance genetics exemplified by sugarcane mosaic virus resistance in maize. Dissertation. Hohenheim University.

Ratcliff, F., B. D. Harrison, and D. C. Baulcombe. 1997. A similarity between viral defense and gene silencing in plants. Science 276: 1558-60.

199

Redinbaugh, MG, R. Louie, P. Ngwira, R. Edema, DT Gordon, and DM Bisaro. 2001. Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation. Journal of Virological Methods 98: 135-43.

Redinbaugh, M. G., W. Houghton, R. Salomon, R. Creamer, S. A. Hogenhout, D. T. Gordon, J. Ackerman, et al. 2002. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathology 92: 1167-74.

Redinbaugh, M. G., R. E. Gingery, and M. W. Jones. 2004. The genetics of virus resistance in maize (Zea mays L.). Maydica 49 : 183-90.

Redinbaugh, M., and R. C. Pratt. 2009. Virus resistance. In Handbook of maize: Its biology., eds. J. L. Bennetzen, S. C. Hake, 251-268. New York: Springer.

Ribas, Alessandra F., Alberto Cenci, Marie-Christine Combes, Herve Etienne, and Philippe Lashermes. 2011. Organization and molecular evolution of a disease-resistance gene cluster in coffee trees. Bmc Genomics 12: 240.

Roane, C. W., C. F. Genter, and S. A. Tolin. 1983. Inheritance of resistance to maize dwarf mosaic virus in maize inbred line Oh7B. Phytopathology 73: 845-50.

Robaglia, C., and C. Caranta. 2006. Translation initiation factors: A weak link in plant RNA virus infection. Trends in Plant Science 11: 40-5.

Rose, John K., and Michael A. Whitt. 2001. Rhabdoviridae: The viruses and their replication. In Fundamental virology., eds. D. M. Knipe, P. M. Howley. Fourth ed., 665- 688. Philadelphia, PA USA: Lippincott Williams & Wilkins.

Rufener, G. K., A. J. Balducchi, R. P. Mowers, R. C. Pratt, R. Louie, M. D. McMullen, and J. K. Knoke. 1996. Maize chlorotic dwarf virus resistant maize and the producction thereof. Patent USA patent 5,563,316, US Patent filed 1996.

Russell, P. 2002. iGenetics. San Francisco, CA: Pearson Education, Inc.

Scheets, Kay, and Margaret G. Redinbaugh. Infectious cDNA transcripts of maize necrotic streak virus: Infectivity and translational characteristics. Virology 350 : 171-83.

Scherm, H., P. S. Ojiambo, and H. K. Ngugi. 2006. Trends in theoretical plant epidemiology. European Journal of Plant Pathology 115: 61-73.

Shepherd, Dionne N., Tichaona Mangwende, Darren P. Martin, Marion Bezuidenhout, Frederik J. Kloppers, Charlene H. Carolissen, Aderito L. Monjane, Edward P. Rybicki, and Jennifer A. Thomson. 2007. Maize streak virus-resistant transgenic maize: A first for africa. Plant Biotechnology Journal 5: 759-67. 200

Shulka, D. D., C. W. Wardand, and A. A. Brunt. 1994. The potyviridae. Oxon, UK: CAB International.

Shull, G. H. 1908. The composition of a field of maize. Report American Breeders Association 4 : 296-301.

Silveira, F. T., J. R. Moro, H. P. da Silva, J. A. de Oliveira, and D. Original Language Perecin. 2008. Inheritance of the resistance to corn stunt. Pesquisa Agropecuária Brasileira. 43 : 1717-24.

Simon, Matthieu, Olivier Loudet, Stephanie Durand, Aurelie Berard, Dominique Brunel, Francois-Xavier Sennesal, Mylene Durand-Tardif, Georges Pelletier, and Christine Camilleri. 2008. Quantitative trait loci mapping in five new large recombinant inbred line populations of arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers. Genetics 178: 2253-64.

Slykhuis, J. T. 1955. Aceria tulipae keifer (acarina: Eriophyidae) in relation to spread of wheat streak mosaic virus. Phytopathology 45 : 116-128.

Smith, C. Wayne, Javier Betran, and E. C. A. Runge. 2004. Corn :Origin, history, technology, and production. Wiley series in crop science. Hoboken, N.J.: Wiley.

Stenger, D. C., J. S. Hall, Il-R Choi, and R. French. 1998. Phylogenetic relationships within the family potyviridae: Wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus. Phytopathology 88 : 782-787.

Stewart, Lucy R., Ashraful Haque Md, Mark W. Jones, and Margaret G. Redinbaugh. 2012. Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus. Mol Breeding. DOI 10.1007/s11032-012-9789-5

Sun, Cuixia, Guangming Zhang, Meng Li, Xiaopeng Wang, Guodong Zhang, Yanchen Tian, and Zeli Wang. 2010. Sequence characterized amplified region markers tightly linked to the dwarf mosaic resistance gene mdm1 (t) in maize (Zea mays L.). Euphytica 174: 219-29.

Tarter, J. A., M. M. Goodman, and J. B. Holland. 2004. Recovery of exotic alleles in semiexotic maize inbreds derived from crosses between Latin American accessions and a temperate line. Theoretical and Applied Genetics 109: 609-17.

Thompson, J. R., and F. Garcia-Arenal. 1998. The bundle sheath-phloem interface of cucumis sativus is a boundary to systemic infection by tomato aspermy virus. Molecular Plant-Microbe Interactions 11: 109-14.

201

Thottappilly, G., N. A. Bosqueperez, and H. W. Rossel. 1993. Viruses and virus diseases of maize in tropical africa. Plant Pathology 42: 494-509.

Todd, Jane C., Casey Hoy, Saskia A. Hogenhout, El-Desouky Ammar, and Margaret G. Redinbaugh. 2010. Plant host range and leafhopper transmission of maize fine streak virus. Phytopathology 100: 1138-45.

Toler, R. W., K. F. Harris, A. J. Bockholt, and G. Skinner. 1985. Reactions of maize (Zea mays) accessions to maize rayado fino virus. Plant Disease 69: 56-7.

Truniger, Veronica, Cristina Nieto, Daniel Gonzalez-Ibeas, and Miguel Aranda. 2008. Mechanism of plant eIF4E-mediated resistance against a carmovirus (tombusviridae): Cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant. Plant Journal 56: 716-27.

Tsai, J. H., and B. W. Falk. 1988. Tropical maize pathogens and their associated insect vector. In Advances in disease vectors research., ed. K. F. Harris. Vol. 5, 177-201. New York: Springer - Verlog.

Tsai, Chi-Wei, Michael Goodin, Saskia A. Hogenhout, Sharon Reed, Margaret G. Redinbaugh, and Kristen J. Willie. 2005. Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins. Journal of Virology 79: 5304- 14.

Tsujimoto, Y., T. Numaga, K. Ohshima, M. Yano, R. Ohsawa, D. B. Goto, S. Niato, and M. Ishikawa. 2003. Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1. Embo Journal 22: 335-43.

Uyemoto, J. K., D. L. Bockelman, and L. E. Claflin. 1980. Severe outbreak of corn lethal necrosis disease in kansas. Plant Disease 64: 99-100.

Uzarowska, Anna, Giuseppe Dionisio, Barbara Sarholz, Hans-Peter Piepho, Mingliang Xu, Christina Ronn Ingvardsen, Gerhard Wenze, and Thomas Lubberstedt. 2009. Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (zea mays L.) by expression profiling. BMC Plant Biology 9: 15.

Valdez, Marta, Kenneth Madriz, and Pilar Ramírez. 2004. A method for genetic transformation of maize for resistance to viral diseases. Revista De Biología Tropical 52: 787-93. van der Biezen, E. A., and J. D. G. Jones. 1998. Plant disease-resistance proteins and the gene-for-gene concept. Trends in Biochemical Sciences 23: 454-6.

202 van Eeuwijk, F. Genotype by environment interaction-basics and beyond. In: Plant Breeding: The Arnel R. Hallauer International Symposium. Lamkey and Lee Eds. Blackwell Publishing, Iowa, U.S. pp. 155-170. van Ooijen, J. W., M. P. Boer, R. C. Jansen, and C. Maliepaard. 2002. MapQTL 4.0, software for the calculation of QTL positions on genetic maps. Vol. 4.0. Wageningen, the Netherlands: Plant Research International. van Ooijen, J. W., and R. E. Voorrips. 2001. JoinMap version 3.0, software for the calculation of genetic linkage maps. Vol. 3.0. Wageningen, the Netherlands: Plant Research International.

Vandeplas, Anneleen. 2003. Evaluation of sixty highland elite maize genotypes for resistance to maize rayado fino virus. Dissertation. The Katholieke Universiteit Leuven.

Vasquez, Jose, and Eloy Mora. 2007. Incidence of and yield loss caused by maize rayado fino virus in maize cultivars in ecuador. Euphytica 153: 339-42.

Vision, T. J., D. G. Brown, D. B. Shmoys, R. T. Durrett, and S. D. Tanksley. 2000. Selective mapping: A strategy for optimizing the construction of high-density linkage maps. Genetics 155: 407-20.

Wang, F., Y. S. Zhang, Y. L. Zhuang, G. Z. Qin, and J. R. Zhang. 2007. Molecular mapping of three loci conferring resistance to maize (Zea mays L.) rough dwarf disease. Mol. Plant Breed. 5 : 178-179.

Wang, Gui-Xiang, Yu Chen, Jiu-Ran Zhao, Lin Li, Schuyler S. Korban, Feng-Ge Wang, Jian-Sheng Li, Jin-Rui Dai, and Ming-Liang Xu. 2007. Mapping of defense response gene homologs and their association with resistance loci in maize. Journal of Integrative Plant Biology 49: 1580-98.

Wangai, A. W., M. G. Redinbaugh, Z. M. Kinyua, D. W. Miano, P. K. Leley, M. Kasina, G. Mahuku, K. Scheets, and D. Jeffers. 2012. First reposr of maize chlorotic mottle virus and maize lethal necrosis in kenya. Plant Disease 96 : 1582-1582.

Weber, Hans, Sbine Schultze, and Arthur J. P. Pfitzner. 1993. Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome the tm-2-2 resistance gene in the tomato. Journal of Virology 67: 6432-8.

Weigel, Detlef, and Jane Glazebrook. 2008. Genetic analysis of arabidopsis mutants. CSH Protocols 2008: pdb.top35,pdb.top35.

203

Weiland, John J., and Michael C. Edwards. 2011. Linear-motion tattoo machine and prefabricated needle sets for the delivery of plant viruses by vascular puncture inoculation. European Journal of Plant Pathology 131: 553-8.

Weiler, E. W., P. S. Jourdan, and W. Conrad. 1981. Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid- phase enzyme immunoassay. Planta Planta 153: 561-71.

Welz, H. G., A. Schechert, A. Pernet, K. V. Pixley, and H. H. Geiger. 1998. A gene for resistance to the maize streak virus in the african CIMMYT maize inbred line CML202. Molecular Breeding 4: 147-54.

White, Donald G. 1999. Compendium of corn diseases. Disease compendium series. 3rd ed. St. Paul, Minn.: APS Press, American Phytopathological Society.

Whitham. 1995. The product of the tobacco mosaic-virus resistance gene-N - similarity to toll and the interleukin-1 receptor (vol 78, pg 1101, 1994). Cell 81: 466-.

Williams, Martin M.,II, and Jerald K. Pataky. 2012. Interactions between maize dwarf mosaic and weed interference on sweet corn. Field Crops Research 128: 48-54.

Wise, Roger P., Matthew J. Moscou, Adam J. Bogdanove, and Steven A. Whitham. 2007. Transcript profiling in host-pathogen interactions. Annual Review of Phytopathology 45: 329-69.

Wisser, R. J., Q. Sun, S. H. Hulbert, S. Kresovich, and R. J. Nelson. 2005. Identification and characterization of regions of the rice genome associated with broad-spectrum, quantitative disease resistance. Genetics 169: 2277-93.

Wisser, R. J., R. J. Nelson, and P. Balint-Kurti. 2006. The genetic architecture of disease resistance in maize: A synthesis of published studies. Phytopathology 96: 120-9.

Witsenboer, H., R. V. Kesseli, M. G. Fortin, M. Stanghellini, and R. W. Michelmore. 1995. Sources and genetic-structure of a cluster of genes for resistance to 3 pathogens in lettuce. Theoretical and Applied Genetics 91: 178-88.

Wu, Jian-yu, Jun-qiang Ding, Yan-xiu Du, Yan-bo Xu, and Xue-cai Zhang. 2007. Genetic analysis and molecular mapping of two dominant complementary genes determining resistance to sugarcane mosaic virus in maize. Euphytica 156: 355-64.

Xia, X. C., A. E. Melchinger, L. Kuntze, and T. Lubberstedt. 1999. Quantitative trait loci mapping of resistance to sugarcane mosaic virus in maize. Phytopathology 89: 660-7.

204

Xiao, Wenkai, Jing Zhao, Shengci Fan, Lin Li, Jinrui Dai, and Mingliang Xu. 2007. Mapping of genome-wide resistance gene analogs (RGAs) in maize (Zea mays L.). Theoretical and Applied Genetics 115: 501-8.

Xu, Yunbi, and Jonathan H. Crouch. 2008. Marker-assisted selection in plant breeding: From publications to practice. Crop Science 48: 391-407.

Xu, Z. L., F. Zou, and T. J. Vision. 2005. Improving quantitative trait loci mapping resolution in experimental crosses by the use of genotypically selected samples. Genetics 170: 401-8.

Yamanaka, T., T. Imai, R. Satoh, A. Kawashima, M. Takahashi, K. Tomita, K. Kubota, T. Meshi, S. Naito, and M. Ishikawa. 2002. Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes. Journal of Virology 76: 2491-7.

Young, N. D. 1996. QTL mapping and quantitative disease resistance in plants. Annual Review of Phytopathology 34: 479-501.

Yu, J., E. S. Buckler, M. D. McMullen, and J. B. Holland. 2008. Genetic design and statistical power of nested association mapping in maize. Genetics 178: 539-11.

Zambrano, Jose L. Selective mapping tutorial. 2012. EXtension, The Ohio State University. http://www.extension.org/pages/61700/selective-mapping-tutorial (accessed 11 April 2012).

Zambrano, Jose Luis, M. David Francis, and M. G. Redinbaugh. 2013. Identification of resistance to Maize rayado fino virus in maize inbred lines. Plant Dis. in press .

Zhang, S. H., X. H. Li, Z. H. Wang, M. L. George, D. Jeffers, F. G. Wang, X. D. Liu, M. S. Li, and L. X. Yuan. 2003. QTL mapping for resistance to SCMV in Chinese maize germplasm . Maydica 48 : 307-312.

Zhang, F. Z., C. Wagstaff, A. M. Rae, A. K. Sihota, C. W. Keevil, S. D. Rothwell, G. J. Clarkson, et al. 2007. QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits. Journal of Experimental Botany 58: 1433-49.

Zhang, Zhi-Yong, Feng-Ling Fu, Lin Gou, Han-Guang Wang, and Wan-Chen Li. 2010. RNA interference-based transgenic maize resistant to maize dwarf mosaic virus. Journal of Plant Biology 53: 297-305.

Zivanovic, Tomislav, Gordana Brankovic, Miroslav Zoric, Gordana Surlan Momirovic, Snezana Jankovic, Sanja Vasiljevic, and Jovan Pavlov. 2012. Effect of recombination in 205 the maize breeding population with exotic germplasm on the yield stability. Euphytica 185: 407-17.

Zwonitzer, John C., Michael D. McMullen, Richard C. Pratt, Peter Balint-Kurti, James B. Holland, Nathan D. Coles, Matthew D. Krakowsky, and Consuelo Arellano. 2010. Mapping resistance quantitative trait loci for three foliar diseases in a maize recombinant inbred line population-evidence for multiple disease resistance. Phytopathology 100: 72- 9.

206