Genetic Analysis and Dissection of Crown Rust Resistance in Diploid Avena Gong-Xin Yu Iowa State University

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1999 Genetic analysis and dissection of crown rust resistance in diploid Avena Gong-Xin Yu Iowa State University

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Genetic analysis and dissection of crown rust resistance in diploid AvsAia

Gong-Xin Yu

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Genetics

Major Professor: Roger P. Wise

Iowa State University

Ames, Iowa

1999

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii

Graduate College

Iowa State University

This is to certify that the Doctoral dissertation of

Gong-Xin Yu

has met the dissertation requirements of Iowa State University

Signature was redacted for privacy.

^Major Professor

Signature was redacted for privacy.

For the Major Program

Signature was redacted for privacy.

For the Graduate College iii

TABLE OF CONTENTS

CHAPTER 1. GENERAL INTRODUCTION 1 Overview 1 Dissertation Organization 2 Literature Review 3 References Cited 11

CHAPTER 2. COMPARATIVE MAPPING OF HOMOEOLOGOUS GROUP 1 REGIONS AND GENES FOR RESISTANCE TO OBLIGATE BIOTROPHS IN AVENA, HORDEUM, AND ZEA MAYS 19 Abstract 19 Introduction 20 Materials and Methods 22 Results 26 Discussion 36 Acknowledgments 41 References Cited 41

CHAPTER 3. AFLP-DERIVED MARKERS FLANKING HYE TIGHTLY-LINKED GENES AT THE PCA (CROWN RUST) RESISTANCE CLUSTER IN DIPLOID AVENA 49 Abstract 49 Introduction 50 Results 52 Discussion 66 Materials and Methods 70 References Cited 74

CHAPTER 4. AN ANCHORED AFLP AND RETROTRANSPOSON-BASED MAP OF DIPLOID AVENA 82 Abstract 82 Introduction 83 Materials and Methods 85 Results 88 Discussion 120 References Cited 123 iv

CHAPTER 5, IDENTIHCATION OF RDH AND RIH, TWO LOCI MEDIATING HYPERSENSITIVE LESION DEVELOPMENT IN RESPONSE TO INFECTION BY THE OAT CROWN-RUST PATHOGEN, PUCCINIA CORONATA F. SP. AVENAE 131 Abstract 131 Introduction 132 Materials and Methods 135 Results 137 Discussion 148 References Cited 149

CHAPTER 6. GENERAL CONCLUSION 155 Conclusions 155 Recommendations for Future Research 157 References Cited 158

ACKNOWLEDGEMENTS 161 1

CHAPTER 1. GENERAL INTRODUCTION

Overview

Avena spp. comprises an important cereal crop for world food production. However, genetic research on Avena has lagged behind that of rice, maize, barley, and other well studied cereal-crop species. Comparative mapping, studies focused on genome comparison by cross-species mapping, has revealed a high level of conservation of gene content and gene orders within related species. If precise colinearity could be established between Avena and those well-studied crop species, extrapolation of information from those crops to Avena would greatly accelerate Avena genetic research.

Plant disease-resistance genes have been recently isolated from a variety of plant species.

These genes condition interactions with a broad range of pathogens, including bacteria, fungi, viruses, and nematodes. Interestingly, all these genes encode proteins that share conserved amino acid motifs. One of the important questions about evolution of these resistance genes is if they are inherited from a common ancestor or are they orthologs.

Comparative mapping focused on resistance gene regions would provide clue to answer this question. If the cross-species mapping could detect conserved linkage between heterologous

DNA markers and the resistance genes, it could be predicted that these genes come from a common ancestor and utilize similar genetic mechanisms against disease pathogens.

Plant resistance genes are commonly organized as clusters in plant genomes. These clusters presumably provide a selective advantage by pyramiding numerous specificities that will protect a host individual against many parasite genotypes, and benefit the host species by providing a reservoir of genetic materials from which new specificities can evolve. About 20 plant disease resistance genes have been cloned and molecularly characterized (Michelmore

1995; Lawton 1997; Michelmore et el. 1998). The mechanisms for recognizing the isolates 2

of plant pathogens and evolving new resistance specificity still remain unresolved (Anderson

et al. 1997; Thomas et al. 1997; Meyers et al. 1998; Ellis et al 1999). Puccinia coronata f.

sp. Avenae, the causal agent of crown rust, is the most important fungal pathogen of

cultivated oat. Pea is a crown rust resistance locus confers multiple specificity to Puccinia

coronata in diploid Avena. Recombination breakpoints were postulated within 5-cM cluster

(Rayapati et al. 1994; Wise et al. 1996). Further characterization of this locus by gene

dissection and gene targeting will improve our understanding of gene organization, evolution

of resistance gene specificity within the Pea resistance cluster.

The hypersensitive response (HR) is a typical defense response associated with gene-for-

gene interactions. The HR involves localized plant cell death at the site of infection.

Localized cell death is assumed to deprive the invading pathogen of nutrients, causing

pathogen containment (Heath 1980; Dangl 1995; Staskawicz et al. 1995). If this were the

case, elucidation and understanding of the cell death gene would be of benefit for crop

improvement.

Amplification fragment length polymorphism (AFLP) and retrotransposon-based,

sequence-specific-amplification polymorphism (S-SAP) are two powerful techniques in the

genotyping of plants. The utilization of these techniques facilitates the construction of highly

saturated genetic maps. These maps can be used to target useful genes in diploid Avena,

therefore, making positional cloning a feasible tool in this species.

Dissertation Organization

Four research manuscripts and a general summary foUow this introduction. The first

manuscript (Chapter 2) was published in Genome (39: 155-164, 1996) and describes the result of comparative mapping of homoeologous group 1 regions and genes for resistance to obligate biotrophs in Avena, Hordeum, and Zea mays. The second manuscript (Chapter 3) 3

has been prepared for publication in Molecular Plant-Microbe Interactions. This manuscript

describes the differentiation of the Pea crown-rust resistance cluster by recombination

breakpoint analysis and Pea crown-rust resistance gene targeting by AFLP bulk-segregant

analysis. The third manuscript (Chapter 4) is being prepared for publication in Genome.

This manuscript describes the establishment of a highly saturated map in a RI population by

AFLP and BARE-l-like retrotransposon-based S-SAP analysis. The fourth manuscript is

being prepared for publication in Molecular Plant-Microbe Interactions. This manuscript

describes the results on a study of inheritance of localized cell death induced by fungal

infection.

Literature Review

Comparative genomic mapping

Comparative genomic mapping has shown an extensive conservation of gene content and

gene order among related plant species. Rice, wheat, barley, rye and maize diverged from a common ancestor about 60 million years ago but share a significant level of marker conservation (Whitkus et al. 1992; Ahn et al. 1993; Ahn and Tanksley 1993; Devos et al.

1993, 1994; Laurie et al. 1993). Evidence for such cross-species gene conservation also comes from comparative studies hetv/een Arabidopsis and oilseed rape. The basic

Arabidopsis gene set is triplicated in the diploid Brassiea crops (Lagercrantz and Lydiate

1996). The triplicated regions of similar genetic length correspond with almost precise colinearity to segments of Arabidopsis that carry major flowering time genes (Lagercrantz et al. 1996;Cavelletal. 1998).

Genomes of modem cereals differ substantially in size. Genome of hexaploid wheat consists of 17,000 Mb per haploid genome while the rice genome (400 Mbp) is 40 fold smaller. Arabidopsis (145 Mbp) is about 117 fold smaller. The difference has been shown to 4 be due primarily to the amount of repetitive DNA sequences and not gene content or gene order (Berhan et al. 1993). Approximately 80% of the wheat genome are the randomly arranged repetitive sequences. In contrast, rice has only 50% of repetitive DNA in its genome. In addition to a high amount of repetitive sequences, the large genome cereals have a much larger ratio of physical to genetic distance (Mb/cM). Physical mapping indicated that barley has an average of 1.5 Mb/cM in contrast to 260 kb/cM in rice and 100-150 kb/cM in

Arabidopsis (Wu and Tanksley 1993; DeScenzo and Wise 1996).

To understand the functions of a gene and explore its potential applications in agriculture, this gene has to be cloned by chromosome walking or any other genomic approaches. The large amount of repetitive sequences and high ratio of physical to genetic distance in the large-genome plants present tremendous obstacles. Conserved gene order among a variety of plant species could have a significant effect on this research. Species with small genomes such as rice and Arabidopsis could be used as a model species to study genes from monocots and dicots (Moore 1993; Foote et al. 1997; Kilian et al. 1997). To be able to do chromosomal walking on the model crops to isolate genes for large genome crops, maps of high resolution must be established. Heterologous DNA markers, which are tightly linked with target genes, can be used as probes to screen YAC or BAG libraries made from genomic

DNA of model crops. After genomic clones are selected, ends of the clones can be isolated and sequenced. PGR primers specific to the sequences, if they could be mapped close to the target gene, could be used to screen the genomic library again. Several rounds of library screening and mapping would eventually detect markers that are right on the genes. This process will accelerate the gene cloning in the large genome crops in that chromosome walking in small genomes is more likely to avoid repetitive sequences. This strategy has been using for the isolation of the wheat chromosome pairing controlling gene, Ph (Foote et al. 1997) and barley stem rust resistance gene, Rpgl (Kilian et al. 1997) by map-based 5 cloning in rice. Although both walks are not yet successful, precise colinearity has been observed over most of the corresponding regions (Foote et al. 1997; Kilian et al. 1997).

In addition to map-based cloning on large genomes by walking on smaller genome model species, comparative genomic mapping has paved a way for cross-species information exchange. Due to conservation of gene order and gene content, data from maize, rice,

Arabidopsis and other well-studied plant species can also be used direcdy or as references in their respective, related species. Another main application of comparative genomic mapping is in determining orthologous relationship of genes among different species. If the cross- species mapping could detect precise colinearity between a common set of heterologous

DNA markers and the target genes, it would be reasonably assumed that those genes were orthologs. If this were true for plant resistance genes, it could be very significant in that resistance genes cloned from one species can be utilized for resistance breeding of another species because they may share a common mechanism against disease pathogens.

Avena is poorly studied and has large genomes, about 11,315 Mb per haploid genome for hexaploid oat and about 3,770 Mb per haploid genome for diploid oat. To use the large amount of available data from other plant species and to determine the orthological relationship among genes in different species, detailed syntenic relationship between oat and other species has to be established. This study would provide a solid base for the improvement of Avena genetics researches.

Resistance gene clusters

Disease resistance genes provide an effective way to prevent disease. Classical studies have demonstrated that resistance genes tend to be clustered in their respective genomes.

These clusters included the L locus and M loci in flax (Linum usitatissimim) (Pryor 1987; and Islam and Shepherd 1991), the Mia locus in barley (Hordeum vulgare L.) (Giese et al.

1981; Wise and Ellingboe 1985), and the Rpl locus in maize (Zea mays) (Hulbert and 6

Bennetzen 1991). Resistance gene clusters are able to confer resistance to many specific strains of pathogens. They are assumed to be a rich resource of genetic diversity in generation of new specificities. A variety of models have been proposed to explain the role of genetic diversity in generating new specificity (Ellis et al. 1999; Hu and Hulbert 1994;

Lawton 1997; Michelmore 1995; Richter et al. 1995; Sudupak et al. 1993). The detailed mechanisms by which novel specificity evolve, however, remains to be understood

(Anderson et al. 1997; Thomas et al. 1997).

Crown rust, caused by fungal pathogen, P. coronata, is one of the most widespread and destructive diseases of cultivated oat. This disease can cause grain yield losses of up to 30% in susceptible oat cultivars (Endo & Boewe, 1958; Frey et al 1973). Host resistance is an important approach to minimize damage caused by pathogen. At present, more than 80 Pc genes have been identified in A. sativa cultivars (Marshall and Shaner 1992). Genetic studies of oat crown-rust resistance indicated that allele for resistance is dominant at most Pc loci, but at some loci, such as Pc-56, Pc-64 and Pc-65 from A. sterilis (Kiehn et al. 1976; Wong et al. 1983), resistance is partially dominant Genes that confer resistance at Pc-12, Pc-13c and

Pc-13d are recessive. Six of the Pc loci are described as complex loci that contain multiple alleles for resistance: Pc-2, Pc-3, Pc-4, Pc-6, Pc-9, and Pc-13 (Simons et al. 1978). Yet littie is known about their organization (Marshall and Shaner, 1992).

Pea is a locus which confer multiple resistance specificities to the crown rust pathogen,

P. coronata, (Wise et al. 1996). This locus was initially identified in a diploid Avena F2 population (Rayapati et al. 1994) and further characterized in a F8.9 recombinant inbred (RI) population developed from the original F2 (Wise et al. 1996). However, genes for individual specificities have not yet been determined. Recombination analysis within the Pea resistance locus would unequivocally identify recombination breakpoints and their order within the 7

locus. Therefore, this research will help to illustrate the genetic organization at this

resistance locus and facilitate its application in the oat breeding for crown rust resistance.

Identifying molecular markers tightly linked to the resistance gene cluster is very

important for both basic and applied applications (Tanksley et al. 1995; Pryor and Ellis

1993). One of the immediate effects is using those markers to study the evolution of

resistance genes. Flanking markers can track unusual recombination events involved in the

resistance gene cluster (Mayo and Shepherd 1980; Hulbert and Bennetzen 1991). At the

/?/7ilocus, evidence was found that the instability in com was associated with recombination and suggested the involvement of unequal crossovers (Sudupak et al. 1993). Resistance gene

tightly linked markers also provided the tool to differentiate different genetic processes involved in generating of rare, non-mendelian, susceptible progeny or spontaneous instability at resistance loci. By this approach, possible genetic events were detected (Wise and

Ellingboe 1985), unequal crossovers (Sudupak et al, 1993), intragenic recombination, or spontaneous mutation. These data will improve our understanding of mechanisms in which genetic diversity is generated and maintained within resistance gene clusters.

DNA markers can efficiently track the transmission of individual resistance alleles if these markers are tightly linked to the resistance genes. Therefore, the tightly linked markers can be very useful in the marker-assisted plant breeding for disease resistance. They allow pyramiding of genes that are effective against all known variants of a pathogen. In plant resistant breeding program, it is often impossible to combine multiple resistances into single genotype using disease screening because of the lack of an isolate diagnostic for each gene.

In contrast, DNA markers have ability to identify which accessions are likely to carry different genes. Furthermore, because resistance genes to diverse pathogens were frequently clustered in the genomes, there is the danger of introducing susceptibility to one disease 8 while breeding for resistance to another (Zeven et al. 1983). This can be averted if the map positions of the different resistance genes were known.

Genetic mapping

A genetic map of an animal or plant species is an abstract model of linear arrangement of a group of genes or markers. Its establishment is usually based on recombination during meiosis. The establishment of genetic map has both theoretical and applied applications. In theory, a genetic map shows the order of its genes, cytogenetical, and any other markers, which represents the current status of a genome. Maps based on conmion set of markers constitute a basis for genomic comparison and, thus, illustrate how genomes evolved

(Tanksley et al 1992; Devos et al. 1992). One of the main applied utilization of the genetic maps is to locate markers that are tighdy linked to genes of interest, either single genes or quantitatively inherited characters. Those markers are especially important for map-based gene cloning and marker assisted breeding.

Three genetic maps have been established for Avena (O'Donoughue et al. 1992; Rayapati et al. 1994 and O'Donoughue et al. 1995). The fust Avena genetic map, published in 1992, consists of 348 loci RFLP markers (O'Donoughue et al. 1992). This map was established based on a population consisted of 44 F2 derived F3 families from a cross between two A- genome diploid species, A. atlantica Baum et Fedak and A. hirtula Lag. The second genetic map for diploid oat was published in 1994 (Rayapati et al. 1994). Two hundred and eight

RFLP markers and 1 locus {Pea) conferring resistance to Puccinia coronata were assigned to ten linkage groups (A-G, L, M, and O). This map was based on a A. strigosa (C.I. 3815) x A. wiestii (CI 1994) F2 population. It consisted of 89 Fj individuals (Rayapati et al. 1994). The third genetic map was published in 1995 for cultivated oats based on cross Avena byzantina

(Koch) cv. Kanota x A. sativa L. cv. Ogle (O'Donoughue et al. 1995). Five hundred and thirty two loci were assigned to 38 linkage groups. These maps established the foundations 9

for the Avena genetic research in that use of these maps can locate markers linked to genes of

interest, both single genes and quantitatively inherited characters. However, genetic maps of

higher resolution are necessary for accurately gene-targeting and map-based gene cloning.

AFLP, recendy developed by Zabeau and Vos 1993, is a novel DNA fingerprinting

technique. The powerfulness of this technique has akeady demonstrated in several cases

(Vos et al. 1995; Becker et al. 1995; Thomas et al. 1995; Biischges et al. 1997). Like AFLP,

S-SAP is also a high throughput technique in plant genotyping. It combines the power of

AFLP and the characteristics of eukaryotic retrotransposon and has been exploited in

developing a multiplex DNA based marker system (Kumar 1996 and Waugh et al. 1997).

This element has a considerable degree of heterogeneity and widespread distribution in plant

genomes, tiierefore, guaranteeing a rich source of DNA markers.

The utilization of retrotransposon-based S-SAP technique has additional effect on

research of genome evolution. Retrotransposons are the most common class of eukaryotic

retrotransposable elements and have long been considered as one main force involved in the

evolution of plant genomes and gene regulation (Flavell et al. 1992; Voytas et al. 1992;

Lindauer et al. 1993; Kumar 1996; Waugh et al. 1997). For example, the promoter regions of

some plant genes were found containing relics of retrotransposon insertions that contribute

transcriptional regulatory sequences (White et al. 1994). It also generates gene duplications:

repetitive retrotransposon sequences provide substrates for unequal crossovers, such event is

thought to have caused zein gene duplication (White et al. 1994). However, it is often

difficult to analyze the activities of the retrotransposon in the genomes and their function due

to the low transposition frequency. Retrotransposon-based S-SAP technique is a potential

tool to analyze the activities of the retrotransposon and their biological effects on the evolution of genomes because of its ability to track the activity of the retroti*ansposons in

genemes. If active retrotransposons increased the frequency of polymorphism, the 10

polymorphism caused by recent insertions and transpositions would be detected as novel

amplified DNA fragments and will inherits as non-Mendelian traits in the mapping

population.

Genetics of fungal-induced hypersensitive lesion development

Localized cell death is a nearly ubiquitous feature of the incompatible interaction of

plants and microbe pathogens. This reaction was termed the hypersensitive response (HR).

This suicide response of infected cells and immediately surrounding tissues is thought to prevent the spreading of pathogen and appeared to be often coupled with resistance phenotypes (Heath 1980; Dangl 1995; Staskawicz et al. 1995). Accumulated evidence indicated that localized cell death is a well-controlled genetic process, similar to programmed cell death (PCD) in animal cells (Doke et al. 1983, 1988; Walbot et al. 1983; Johai et al.

1995; Baker and Olandi 1995; Low and Merida 1996). However, the role of the localized cell death in the development of plant disease resistance is less conclusive. The contribution of cell death in plant defense against pathogen attack is evident in case of Arabidopsis Isd lesion mutants. These mutants induced local and systemic resistance to varieties of pathogens even in susceptible plant lines (Dietrich et al. 1994; and Jones 1994). In contrast, disease resistance could happen without the localized cell death. These include the mlo- mediated barley resistance against all races of Erysiphe graminis f. sp. hordei

(Freialdenhoven et al. 1996) and i?;c-mediated resistance to potato virus X (Kohm et al.

1993). Therefore, further research is necessary to study the localized cell death for understanding the mechanisms of the localized cell death and its role in the development of the plant disease resistance. 11

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CHAPTER 2. COMPARATIVE MAPPING OF HOMOEOLOGOUS GROUP 1

REGIONS AND GENES FOR RESISTANCE

TO OBLIGATE BIOTROPHS IN AVENA, HORDEUM, AND ZEA MAYS

A paper published in Genome

Gong-Xin Yu''^ Aria L. Bush^"^, and Roger P. Wise

Abstract

The colinearity of markers linked with resistance loci on linkage group A of diploid oat,

on the homoeologous groups in hexaploid oat, barley chromosome IH, and on homoeologous

maize chromosomes were determined. Thirty-two DNA probes from homoeologous group 1 chromosomes of the Gramineae were tested. Most of the heterologous probes detected

polymorphisms that mapped to linkage group A of diploid oat, two linkage groups of

hexaploid oat, barley chromosome IH, and maize chromosomes 3, 6, and 8. Many of these

DNA markers appeared to have conserved Unkage relationships with resistance and prolamin loci in Avena, Hordeum, and Zea mays. These resistance loci included the Pea crown rust resistance cluster in diploid oat, the R203 crown rust resistance locus in hexaploid oat, the

Mia powdery mildew resistance cluster in barley, and the rp3, wsml, wsm2, mdml, ht2, and htnl resistance loci in maize. Prolamin encoding loci included Avn in diploid oat and Horl and Horl in barley. A high degree of colinearity was revealed among the common RFLP markers on the small chromosome fragments among these homoeologous groups.

'interdepartmental Genetics Program, ^Department of Plant Pathology, ^Com Insects and Crop Genetics Research, U.S. Department of Agriculture-Agricultural Research Service, (USDA-ARS), Iowa State University, Ames, lA 50011-1020, U.S.A. 20

Introduction

Comparative mapping displays a broad range of colinearity among related species.

However, research in this area only has just begun to address agronomically important genes.

Genes for host plant resistance to obligate biotrophs are involved in recognition of invading pathogens and activation of defense systems. They have been used in crop breeding programs because of their effectiveness in preventing disease and ease of handling as single

Mendelian loci. Lack of knowledge about the products of resistance genes has made their molecular isolation difficult (Martin et al. 1993). Another difficulty is presented by the large amount of repetitive sequences in plant genomes, especially those of agriculturally important species like oat, wheat and barley (Moore et al. 1993; Tanksley et al. 1995). These obstacles have hindered efforts to examine the evolution of resistance genes and their role in recognition and defense against specific pathogens.

Comparative genomic mapping, using a common set of restriction fragment length polymorphism (RFLP) markers on several different species, has proven to be a powerful tool to address these problems (Bennetzen and Freeling 1993; Helentjaris 1993; Moore et al.

1993). Several studies have demonstrated a significant level of marker conservation among related species (Whitkus et al. 1992; Ahn et al. 1993; Ahn and Tanksley 1993; Berhan et al.

1993; Devos et al. 1993, 1994). Tanksley et al. (1992) constructed a high density comparative RFLP map using DNA probes common to tomato and potato and found that the genomes are nearly identical in overall gene content and gene arrangements. Ahn and

Tanksley (1993) also noted that conservation exists between the chromosome regions belonging to the different Poaceae tribes of wheat, maize, and rice, especially within the short fragments of chromosomes. The difference between maize and sorghum genomes has been shown to be due primarily to the amount of repetitive DNA sequences and not gene content or gene order (Berhan et al. 1993). Moore et al.(1993), therefore, proposed that rice. 21 a small genome species, may be used as a model to isolate genes from monocots and study gene function. Additionally, rice clones could be used to jump and walk between markers in large genome species to isolate specific genes, providing that marker colinearity can be shown. Although many studies have examined the colinearity among different species

(Wang et al. 1992; Whitkus et al. 1992; Ahn et al. 1993; Ahn and Tanksley 1993; Berhan et al. 1993; Devos et al. 1993, 1994), few have investigated specific genes, such as host resistance genes.

The Gramineae family contains approximately 10,000 species including agriculturally important crops such as wheat (Triticum aestivum), rice (jOryza sativa L.), barley (Hordeum vulgare L.), oat (Avena sativa L.) and maize (Zea mays L.). In this family, group 1 chromosomes have been studied extensively because of many important genes mapped on them (Van Deynze et al. 1995). Among the group 1 chromosomes, linkage group A of diploid oat contains Pea, a cluster of loci conferring resistance to the crown rust fungus,

Puccinia coronata cda f. sp. Avena Eriks, and Avn, a prolamin seed-storage protein gene

(Rayapati et al. 1994a, 1994b). Genes encoding prolamins are often interspersed with different fungal resistance genes (Wettstein-Knowles 1990; Howes et al. 1992; Wang et al.

1992) although this has not been shown in diploid oat. The hexaploid oat line D494 contains

R203, a crown rust resistance locus linked to several RFLP markers on linkage group A in diploid oat (Bush et al. 1994). Recently, the prolamin locus Avn-C has also been positioned on homoeologous group A near the same RFLP markers (O'Donoughue et al. 1995).

Similarly, barley chromosome IH has several linked Ml genes determining resistance to the powdery mildew fungus, Erysiphe graminis f. sp. hordei (Jorgensen and Moseman 1972;

Giese et al. 1981; Gorg et al. 1993). Recently, a high resolution map has been established on the short arm of barley IH that contains the Mia resistance-gene cluster flanked by the prolamin-encoding genes Horl and Hor2 (DeScenzo et al. 1994; Mahadevappa et al. 1994). 22

Several genes for resistance have been placed on the maize genetic map (Coe 1993), including rp3 conferring resistance to maize common rust, caused by Puccinia sorghi

(Wilkinson and Hooker 1968), wsml and wsm2 conferring resistance to wheat streak mosaic virus (McMullen et al. 1994), mdml (Simcox and Bennetzen 1995) conferring resistance to maize dwarf mosaic virus, and ht2 and htnl (Simcox et al. 1993) conferring resistance to

Helminthosporium maydis, and Setosphaeria turcica, respectively.

In this report, we present a comparative study of homoeologous group 1 chromosome fragments carrying resistance loci in diploid and hexaploid oat, barley, and maize.

Heterologous DNA probes were used to determine the colinearity of the resistance loci and markers linked to them.

Materials and Methods

Genetic stocks

The mapping populations used in this study are as follows.

1. The diploid Avena population was generated by crossing A. strigosa (CI 3815) with A. wiestii (CI 1994) (Rayapati et al. 1994b). A. strigosa is resistant to 40 isolates of P. coronata in the ISU collection whereas A. wiestii is susceptible to the same isolates (Wise and Gobelman-Wemer 1993). One hundred F8:9 recombinant inbred (RI) lines generated by single seed descent from an original F2 mapping population was provided by Dr. M. Lee

(Iowa State University). The Pea resistance cluster, conferring resistance to nine isolates of

P. coronata, was positioned on linkage group A in the original F2 population (Rayapati et al.

1994a, 1994b).

2. The hexaploid Avena sativa population was generated by backcrossing the D494

(PI501536) rust resistant line (Prey et al. 1988) to the susceptible recurrent parent Lang.

D494 and Lang display differential reactions to at least 6 isolates of P. coronata in the ISU 23 collection. Resistance to isolate 203 was mapped with respect to several RFLP markers

(Bush et al. 1994). One hundred BC1F2 plants originated from a single BCiFi plant were used to determine linkage between RFLP markers and the rust resistance locus.

3. The Hordeum vulgare IHS interval mapping population consists of 30 lines, each containing a reciprocal recombination event between mapped markers on chromosome IH.

These characterized individuals were selected from 285 recombinant lines derived from a cross of CI 16151 (containing Mla6 mAMlal4 ) and CI 16155 (containing Mlal3). This population was used previously to construct a saturated linkage map in the region flanking the Mia powdery mildew resistance gene cluster on chromosome IH (DeScenzo et al. 1994;

Mahadevappa et al. 1994).

4. Two Zea mays populations, one consisting of 48 RI lines from the cross of T232 and

CM37, another consisting of 38 RI lines from the cross C0159 and Tx303 were provided by

Dr. B. Burr at Brookhaven National Laboratory, U.S.A. (Burr et el. 1988).

Rust inoculation

To position the Fca crown rust resistance locus (Rayapati et al. 1994b), an ISU isolate of race 290 was used to inoculate eight individuals from each F8:9 family from the diploid oat population as described previously (Bush et al. 1994). Disease development was assessed at

10 days after inoculation on a 0 to 4 scale using the method of Murphy (1935), where infection types (IT) of 0,1 and 2 are considered resistant, and 3 and 4 are susceptible. Oat cultivar 'Markton' was included as a susceptible control. An isolate of race 203 was used to position the R203 locus in BC1F2 plants derived from D494 x Lang hexaploid oat (Bush et al. 1994).

DNA extraction, Southern blotting, and hybridization

Fresh leaf tissue samples (0.5 gram) were harvested from greenhouse grown plants and frozen at -8OOC. Restriction fragment length polymorphism analysis was performed as 24

described previously (Bush et al. 1994; Wise and Schnable 1994). The stringency of post-

hybridization washes was modified because of the lower Tm associated with some of the

heterologous DNA probes. The membranes were processed 4 x 30 min at 65°C in 1 x SSPE

(20X SSPE = 3.6 M NaCl, 0.2 M NaH2P04, 20 mM Na2EDTA, pH 7.4), 0.1 % SDS and

exposed to Kodak X-Omat AR film for 3-5 days at -80°C with one or two intensifying

screens.

Parental DNAs (10|ig per lane for diploid oat, barley, or maize and 20 |ig per lane for

hexaploid oat) were assayed for polymorphisms after digestion with EcoRV, Kpnl, HindllL,

Sad, Dral, EcdRl and BglW in separate reactions and transferred to Hybond N+

(Amersham). Thirty-two DNA probes were used to screen the parental DNAs (Table 1).

Based on the results of the parental survey, appropriate probe-enzyme-population combinations were selected to optimize the number of polymorphisms that could be screened.

Data analysis

Polymorphic DNA bands detected by a single probe was scored separately. If more than one polymorphic band was present, bands were labeled by a letter suffix to the probe name, in descending size order. If codominant alleles could be identified, the locus was labeled in the same manner. Upper case letters were used for polymorphic oat and maize loci, while numbers were used for those in barley, in accordance with current standards of nomenclature for each species.

Mapping hexaploid and diploid oat markers was performed as follows. Data were entered into Map Manager V2.6 (Manly 1993) and further prepared for MapMaker by using the 'Export for MapMaker' command prior to mapping in MapMaker 3.0 (copyright 1993,

E.I. Dupont de Nemours and Co., Lander et al. 1987). All map distances were calculated in

MapMaker using the Kosambi correction. In case of hexaploid oat, data were coded as an ¥2 25

Table 1. The type and source of DNA probes used for determination of homoeoiogous

groups in the Gramineae.

Number Probe used Type Source ISU 10 oat root cDNA M. Lee, Iowa St. Univ. CDO 5 oat leaf cDNA Sorrells and Tanksley, Cornell BCD 2 barley cDNA Sorrells and Tanksley, Cornell ABC 1 barley cDNA Kudma and Kleinhofs, Wash. St. Univ. Chsl 1 barley cDNA W. Rohde, Max-Planck-Institut. Hor 2 barley cDNA B. Ford, Rothamsted Exp. Station ABO 2 barley genomic Kudma and Kleinhofs, Wash. St. Univ MWG 5 barley genomic A. Graner, Griinbach crwsio 1 barley cloned RAPD S. Somerville, Carnegie Inst. Washington. KSU 2 wheat genomic B. Gill, Kansas St. Univ. WG 1 wheat genomic Sorrells and Tanksley, Cornell

intercross. Markers were grouped into subsets at a LOD of 6.0, and distantly linked markers

were placed with a LOD of 3.0. The diploid oat data were coded for RI self analysis, and grouped into subsets with a LOD cutoff of 6.0. Subsets were linked and arranged relative to each other using a LOD of 2.5. Markers were integrated into the barley interval population

by importing the data directly into our existing Map Manager data base. Data from the maize populations were integrated into the Brookhaven maize RFLP map by Dr. B. Burr,

Brookhaven National Laboratory. Suffixes of letters A-G identify markers found either in the T232 x CM37 population alone or both populations if a locus from the two populations was merged. The suffixes of letters H-L were used for the C0159 x Tx303 population only.

Pertinent map scores were downloaded from the Maize Database (World Wide Web) and imported directly into our Map Manager files to check for crossovers to confirm the order of 26 closely linked markers on maize chromosomes 3,6,and 8.

Results

RFLP survey of parents of the mapping populations

Thirty two heterologous probes were examined. Conserved DNA sequences in the oat, barley, and maize genomes were identified with most of the heterologous DNA probes. The number of bands and the strength of hybridization signals are shown in Table 2. Sixty percent of the heterologous probes revealed visible hybridization signals in oat, 80% in barley and 70% in maize. However, a broad range of sequence differentiation was demonstrated in these hybridizations. As shown in Table 2, when fifteen oat cDNA probes were used to hybridize to barley and maize, six probes showed very strong hybridization signals that are comparable to those on oat. Three probes did not hybridize to barley or maize at all. The remaining five probes hybridized weakly. A similar range of signals were seen when oat and maize were hybridized with barley cDNA or genomic probes.

Table 2 also shows that the difference in the number of polymorphisms detected by heterologous probes in oat, barley, and maize. The four maize accessions displayed more genetic diversity than oat or barley. Of 22 probes that identified conserved sequences in maize, 18 detected polymorphisms. In contrast, only 6 of the 19 probes previously mapped on barley IH detected polymorphisms in both diploid and hexaploid oat, and 3 of the 13 probes from linkage group A in diploid oat detected polymorphisms in barley. The lack ofpolymorphism in barley is not surprising, since the original cross used to develop the mapping population was quite narrow.

Comparison of linkage group A in F2 (selO and F$;9 (RI) progeny of diploid oat

In a previous analysis of the F2 population used to generate the RI population used in this 27

Table 2. Homoeologous group 1 probes tested: signal strength and number of hybridizing restriction fragments

Number of hybridizing

Pr°be restriction fragments^ Signal strength^* 2x oat 6x oat barley maize oat barley maize

ISU1410 10*c 15* 15* 5* N +++++ +++++

CD01473 4* 7* - - N - - CD0708 1* 3 1 2* N +++ ++ CD0580 1* 5 1* 2* N +++++ ++++ ISU2232 2* 4* 1* 5* N ++++ +++++ ISU1774 1* 3* 2 5* N +++++ +++++

CD0685 1 3 1 - N +++++ -

ISU916 1* 2* - - N - - ISU1719 1* 3* 2 4* N + ++++ ISU1146 1* 3* 1 1* N + + ISU2117 4* g* 1 3* N + ++

ISU1785 1* 2* - - N - - ISU2191 11* 16* 9* 7* N +++++ +++++ ISU2192 1* 3* 5 4* N +++++ +++++ CD01519 2 5 1 1 N +++ ++

Hor2 _ _ 12* _ _ N . 28

Table 2. (continued)

MWG645 4 10* 4 18* +++++ N +++++ MWG060 2* 5* 2* 2 + N +

MWG036 - - 3* - - N -

CIWSIO - - 1* 4# - N + BCD249 1 5* 2* 6* + N +++ MWG068 3* 7 2 2 ++ N ++ Chsl 1 4 2* 3* +++++ N ++++ ABG373 1 3 2 1* + N +++

Horl - - 5* - - N -

ABG452 - - 4 - - N - DGE18 1 3 3* 4* +++++ + DGG9 1 3 1 3* +++++ ++++ WG180 2 3 4 1 + + BCD98 1* 4* 2* 7* +++ N +++

MWG075 - - 4* - - N -

ABC160 _ 1* _ +++ N _

^ The number of bands detected on oat, barley and maize DNAs when digested with the restriction enzyme KprH.

^ The relative signal strength in comparison to the signal strength detected by the homologous probe."+" or"++" indicates weak hybridization signals and "++++" or "+++++" strong hybridization signals. indicates no detectable hybridization. N denotes probe originated from the same species. c Columns marked with indicate species-probe combinations that detected polymorphisms. study, 10 oat cDNAs were used to position 10 RFLP loci on linkage group A in diploid oat

(Rayapati et al. 1994b). In the current analysis, we used 9 of the previous oat cDNAs in 29 addition to 3 additional oat cDNAs, 1 barley cDNA, and 2 barley genomic probes to position

21 RFLP loci. Generally, the linear order of loci was consistent between the two populations with minor rearrangements most likely due to filling in gaps with additional markers. The F2 framework order was established at LOD 3.0 or greater, however, the 35.2 cM gap between

Xcdol473A and Xisul 146 in the F8:9 analysis had a LOD of 0.6. Since the probable order had been established previously in the F2 map, we estimated the most likely linear arrangement of the two group 1 linkage groups (Fig. 1). The order of RFLP loci detected by

CD01473, MWG068, and CD0580 was also consistent with results from a diploid mapping population derived from a cross between A. atlantica and A. hirtula, although they positioned a locus detected by BCD98 to an interval at the end of linkage group B (O'Donoughue et al.

1992; Van Deynze et al. 1995). Since oat appears to be prone to translocations

(O'Donoughue et al. 1995), this is not surprising.

Comparison of group 1 linkage groups among hexaploid oat, diploid oat, and barley

The arrangements of DNA markers and their relationships with resistance loci in hexaploid oat, diploid oat, and barley are shown in Fig. 1. In hexaploid oat, two linkage groups were identified. The first linkage group spanned a genetic distance of 21.9 cM including the crown-rust resistance locus R203 and six other loci detected by MWG060,

ISU1719, ISU1774, and ISU1410. The second linkage group consisted of six loci detected by ISU916, ISU2232, MWG060, ISU1774, CD01473 and ISU1719 and covered 27 cM. A seventh locus on the second group was detected by BCD249 was positioned 46.3 cM from

Xisu916. The results suggest that these two linkage groups were homoeologous although the alignments of some common markers were changed.

The alignments of the Xisu916, Xmwg060A, Xisul?19A, and Xcdol473 loci on linkage group A in diploid oat adjacent to Avn were identical to the second linkage group in hexaploid oat. The Pea rust-resistance cluster was distantiy linked to the Avn region on 30 linkage group A in diploid oat (Rayapati et al. 1994a). Although we have not identified a rust isolate that detects a resistance locus in diploid oat corresponding to R203 in hexaploid oat, conservation of marker order between diploid and hexaploid oat in this region suggests that there may be a yet undetected resistance locus positioned here in diploid oat as well.

Conservation with limited groups of markers was also indicated between oat and barley.

Of 19 probes previously mapped on barley chromosome IH, all four of the polymorphic probes were positioned on linkage group A in diploid oat. The linear order detected by

CD0580, ISU2232, ISU1410, and MWG060 on diploid oat was completely conserved in barley, however, it was inverted in comparison to our best estimate for diploid oat. Other common markers were also rearranged between the two group 1 linkage groups, suggesting that oat may have some translocations and rearrangements in comparison to barley (Fig. 1).

Additional markers are needed in the two large gaps in diploid oat to accurately resolve these putative rearrangements, however.

Identification of homoeologoiis linkage groups in maize

Most of the probes from barley and oat detected homoeologous sequences in maize, identifying at least two loci per probe in the maize genome (Table 2). The number of loci and their distribution on the ten maize chromosomes are shown in Table 3. On maize chromosomes 3, 6 and 5, the loci detected by heterologous group 1 probes were almost always concentrated within small chromosome fragments positioned near the centromeres.

On the other seven chromosomes, the loci detected by the heterologous group 1 probes were distributed randomly. The homoeologous groups in maize detected by the group 1 heterologous probes are shown in Fig. 2. The alignments of markers in oat and barley were found to be conserved in maize. On chromosome (5, four loci detected by CD0580,

ISU2232, ISU1410, and BCD98 had the same linear order as those on linkage group A in 31

Table 3. Marker distribution on maize chromosomes

Chromosome Number of RFLP markers^ Total genetic distance (cM) 1 9 166 2 9 236 3 6b + 2C 33.3d 4 4 100 5 2 6 7 22.4 7 6 115 8 12+1 70.3 9 2 10 4 78

^ The number of markers mapped on individual maize chromosomes.

^ The number of markers that were closely linked on maize chromosomes. c The number of markers genetically distant from the closely-linked groups.

The genetic distance spanning closely linked groups. diploid oat and the order of loci detected by CD0580, ISU2232, ISU1410, and MWG645 was conserved on barley chromosome IH. Partial conservation of marker arrangements was also found when the two other linkage groups formed in maize were compared with diploid oat linkage group A and barley chromosome IH. The order of 3 loci detected by 1SU1719,

ISU2117, and ISU2191 on diploid oat linkage group A was found intact on maize chromosome 3. In addition, some linkage relationships found on linkage group A were also found on the homoeologous group on maize chromosome 8. Six disease resistance loci previously mapped on maize chromosomes (Coe 1993; Simcox and Bennetzen 1993;

McMullen et al. 1994; Simcox et al. 1995) were located within the three homoeologous linkage groups; rp3 and mm2 were positioned on chromosome 3, and wsml and mdml on Fig. 1. Comparative maps of diploid oat linkage group A, hexaploid oat, and barley chromosome IH. Shadowed loci correspond to loci conferring resistance or encoding prolamin storage proteins. Note the hexaploid and diploid oat maps are drawn on a different scale than barley. 33

Hexaploid oat Diploid Oat Barley

- - XisuUlOB 1.0 12.7 Xcdo1473B 3.6 Xcdo1473C 1.1 10.4 - - Xmwg068 -y f / :1.1 -- 25.7 0.7 -- XisuUlOA \ / 4.8 -- XisuUIOC Xcdo708 \/ 0.8 4.3 K 7.6 \ XcdoSSO sm \ 9.0 1.3 R20S \ Xisu2232 0.3 XmwgOeOB 10.0 ^ / \ XisuUlOE •>iy N- 0.3 10.8 0.2 Xisu1774 K\ 1.0

XisufTIS \ 1.5 9.0 -- Xcdo1473A \ w If \ \ .2.0 III 35.2 III \ \ -- -- Xbcd249 III \ III -. Xisu1146 \ 3.5 III 14.7 \ III \ 46.3 mjm7 \ III 9.9 \ \ Xisu178S III 6.8 Xisu2191A III 3.8 Xisu2191C 1.5 Xisu2191D -- Xisu916 -'ill 13.3 \ 11.2 , JII Xisu2192 \ \ u 23.5 2.3 == mVi^K J \ -- FC9t 7.2 -- Xcdo1473 J I 10 Fig. 2. The homoeologous linkage groups on maize chromosome 3, 6, and 8 detected with

Gramineae group 1 heterologous DNA probes. Previously mapped markers and their genetic distances are listed on the left side of the maps. Loci detected by heterologous DNA probes from barley and oat are listed on the right. Horizontal lines connecting two markers on both sides of the maps indicate cosegregation. Horizontal lines extending to one side only indicate the degree of linkage of mapped markers. Loci conferring disease resistance are shadowed. 35

Maize 3

i(nPiZ47 J Xbcd98J Xnpi114B 0-^= - Xisu1719H w 5.9 Xisu2117l Maize 8 Xuaz34B - - Xisu1719B, Xisu2191C 5.9 Xumc102 oe- rp$ XumclSA wsmZ Xbnl6.06 Xnpi110 XisuUlOA 10.7 19.5 Xvpl Xmwg645C i 4.6 ~ XbnlS.37 Xucsd64B o o Xbcd98B Xmdhi f ° Xisu1719C. Xisu2191B Xnpi618 l-S Xisu2191H Xumc96 Xlsu1410l 11 17.1 Xbnl7.26 Xmwg64SJ XumcieOB 4.1 Xmwg64SK &1 2.2 Xcdo708 Xufu 2.2 Xcdo580. Xdgg9A Maize 6 19.4

Xisu1774B Nor mdml Xumc48 Xbnl6.29 M wsmf .Jsui 11.6 ht2 Xnpl23S 4.5 Xisu2232H Xbnl7.28 Xumc117 7.6 htnl Xnpl594 -fs^uhn 5.6 Xumc4B Xbcd98E Xenpl WM 36 chromosome 6. ht2 and htnl, conferring resistance to the northern com leaf blight fungus,

Helminthosporium turcicum, were positioned on chromosome 8.

Discussion

Identification of homoeologous relationships among hexaploid oat, diploid oat, barley and maize

In theory, hexaploid oat should have three linkage groups corresponding to diploid oat linkage group A. Two linkage groups were discovered in hexaploid oat that are homoeologous with linkage group A in diploid oat. Analysis of common detectable markers on hexaploid and diploid oat demonstrated that the marker orders were conserved on diploid oat linkage group A and its homoeologous hexaploid linkage groups. The homoeologous linkage in hexaploid oat exhibited nearly perfect colinearity of marker arrangement with the markers near Avn in diploid oat. Similar arrangements of some of these DNA markers and

Avn locus was reported in hexaploid oat by O'Donoughue et al. (1995). Conservation was also indicated between oat and barley although some marker rearrangements occurred, suggesting an inversion between linkage group A from A. strigosa x A. wiestii and barley IH

(Fig. 1). Overall, these results are in agreement with the observation that the group 1 chromosomes of Triticeae species are homoeologous to linkage group A in diploid oat (Van

Deynzeetal. 1995b).

In maize, it appeared that three homoeologous linkage groups were detected instead of the two predicted by the hypothesis that maize is descended from an ancient tetraploid species (Ahn et al. 1993). However, when the three linkage groups were compared in detail, it was found that except for the three highly repetitive probes, ISU1410, BCD98, and

MWG645, almost all of the other probes detected two homoeologous sequences among these linkage groups or in other locations in the maize genome. CD0580 and ISU1774 detected 37

two loci on maize chromosome 6 and 8 while ISU1719 and ISU2191 detected two loci on

chromosome 3 and 6. This result was in agreement with the hypothesis that maize is

descended from an ancient tetraploid species.

The comparative analysis of group 1 probes demonstrated the chromosome fragments of

oat and barley were conserved in maize chromosome 6 and S. There were also many duplicated sequences of these homoeologous groups on maize chromosome S. These results

are in agreement with previous reports that maize chromosomes 3, 6, and 8 shared duplicate sequences (Helentjaris et al. 1988). The locations of these homoeologous linkage groups in maize detected by the probes from linkage group A of diploid oat and chromosome IH of barley are the same as those detected by the probes from rice chromosome 5 (Ahn and

Tanksley 1993). Conserved marker alignments were also detected among the three species.

The marker order detected by CD0580-ISU2232-ISU1410-BCD98 and CD0708-CD0580-

ISU1774 in diploid oat, as well as CD0580-DGG9-MWG645 in barley were maintained on these maize chromosomes.

Conservation of resistance gene-iinked DNA markers in oat, barley and maize

Substantial conservation of gene order was observed within small chromosome fragments of Gramineae family species (Ahn et al. 1993; Devos et al. 1993, 1994; Van Deynze et al.

1995). If group 1 chromosomes among the Gramineae descended from the chromosome of a common ancestor, the disease resistance loci on them may be orthologous. Therefore, the linear order of DNA markers around the resistance loci should be conserved. Conservation of linkage relationships among the common markers was evident among the oat, barley, and maize genomes. Linkage group A of diploid oat, its homoeologous linkage groups in hexaploid oat and IH in barley included disease resistance loci (Bush et al. 1994; DeScenzo et al. 1994; Rayapati et al. 1994b). It was interesting to find that all the three homoeologous linkage groups formed in maize also included resistance loci (Coe 1993; Simcox and 38

Bennetzen 1993; McMullen et al. 1994; Simcox et al. 1995). These results suggest the large extent of linkage conservation of common markers and loci conferring disease resistance among linkage group A in diploid oat, its corresponding hexaploid oat linkage groups, IH in barley, and maize chromosomes 3,6 and 8. However, the resistance loci were not flanked by the same molecular markers, therefore, it was not possible to determine if these loci are orthologous. Interestingly, the loci detected by these probes are centromeric in maize yet they were placed toward the end of linkage groups in oat and barley. This may be due to a series of breakage fusion cycles during the evolution of these chromosomes from a common ancestor (Helentjaris et al. 1988).

Genetic changes in oat and maize

Fig.s 1 and 2 illustrate, that, although there are highly homologous regions in these species, the alignments of some common probes on the maize chromosomes are changed and genetic distances are compressed compared to those observed in oat and barley. For example, loci detected by ISU1719 and ISU2191 are distantly linked on linkage group A in diploid oat, but cosegregate on maize chromosomes 3 and 8. Similar results were found between CD0580 and DGG9, and DGG9 and MWG645 when barley and maize are compared. This variation in marker order and genetic distance was also shown when rice probes were mapped on maize chromosomes (Ahn et al. 1993; Causse et al. 1994).

Deletions, translocations, and inversions may be involved in this process (Ahn and Tanksley

1993). Other factors which inhibit recombination also may be involved such as centromeres, telomeres, heterochromatin, or other highly repetitive sequences (Tanksley et al. 1992).

The importance of documenting the degree of homology on genetic maps

Comparative genomic mapping can be used to obtain information concerning the evolution of plant species (Tanksley et al. 1992; Whitkus et al. 1992; Ahn et al. 1993; Ahn and Tanksley 1993; Devos et al. 1993, 1994; Laurie et al. 1993) and to transfer genetic 39 information from one species to another (Moore et al. 1993). It can also be used to explore the orthologous relationships between important genes and colinearity of marker alignments in the related species. However, the maps currently used are limited to linkage relationships and do not indicate the degree of homology.

As can be seen in Table 2 and Fig. 3, the heterologous DNA probes varied in the extent of homology when they were used to hybridize to oat, barley, and maize, indicating there is range of sequence differentiation during the evolution process. Similar observations have been reported in other related studies (Hulbert et al. 1990; Devos et al. 1993). Different bands detected by the same probe also showed variable strength of hybridization signals. For comparisons among diploid plant species such as oat and barley, the strength of the hybridization signal can delineate the extent of sequence conservation since divergence from a common ancestor. For the comparison of diploid oat, barley, and maize, an ancient tetraploid species, a single-copy locus in diploid oat and barley may be duplicated in maize

(Ahn et al. 1993). As shown in Table 2, of 19 probes which detected single copy loci in diploid oat and barley, twelve (about 60%) detected duplicate sequences on maize, while 7 detected single copies. The single copy loci detected on maize may represent those sequences for which the duplicate counterparts have been lost or have diverged to the extent that they are no longer detectable by hybridization. For duplicate loci that still exist in maize, the biological functions of these duplicate loci (or genes) need to be considered. The sequences of functional genes are conser\'ed more strongly than other sequences on genomes

(Bennetzen et al. 1994). Therefore, if the signal strength of some bands detected by probes is strong, it may represent a homoeologous sequence in maize that may be a functional gene. In contrast, if signal strength is weak, the sequence may have changed to the extent that it has lost its function or obtained a new function. Therefore, it would be prudent to document this additional information to increase the value of comparative genomic maps. 40

Kpn\ Hind\\\

S» ^ wV ^ ^ ^ >s? 0- 0- .N »?S r"^

wl*

Fig. 3. DNA gel blot hybridization of barley genomic probe MWG068 on diploid oat, hexaploid oat, and barley. D494 and Lang are hexaploid oat. C.I. 3815 and C.I. 1994 are diploid oat. Barley accession C.I. 16151 contains teh Mla6 (powdery mildew) resistance gene and C.I. 16155 contains Mlal3. 41

This study revealed the extent of conservation of common markers and resistance loci on diploid linkage group A, the homoeologous linkage groups in hexaploid oat, chromosome IH in barley, and three maize chromosomes in addition to markers flanking prolamin loci in oat and barley. Generally, as in other comparative mapping studies, there was colinearity on small chromosome fragments among these linkage groups. Although the accurate colinearity of the common markers around loci conferring resistance could not be completely determined, a foundation was established to explore the orthological relationships of diese loci in future research.

Acknowledgments

The authors recognize the technical assistance of Mark Brown, Edwards Allen, and

Sophie Franck for DNA preparation and analysis. We would also like to acknowledge the sharing of probes from the plant genetics community. We thank Mike Lee for the diploid oat

RI population, and Ben Burr for the maize RI population in addition to integration of data into the Brookhaven maize RI map. We thank Charlotte Bronson and Allen Miller for review of the manuscript. This research was supported in part by USDA-NRI/CGP grants

AMD 93-37300-8770 and AMD 93-37300-8768. A. L. B. was supported by a USDA-ARS

Postdoctoral Research Associateship.

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CHAPTER 3. AFLP-DERIVED MARKERS FLANKING FIVE TIGHTLY-LINKED

GENES AT THE PCA (CROWN RUST) RESISTANCE CLUSTER IN DIPLOID

AVENA

A paper to be submitted to Molecular Plant-Microbe Interactions

Gong-Xin Yu'*^, and Roger P. Wise'"^'^

Abstract

Puccinia coronata Corda f. sp. avenae Eriks, the causal agent of crown rust, is the most important fungal pathogen of cultivated oat. There are many genes, designated Pc, that provide resistance to this pathogen. The Pea crown rust resistance gene cluster confers specificity to at least eleven isolates of P. coronata. Seven recombinant inbred (RI) lines that contained putative recombination breakpoints within the Pea cluster were backcossed to the susceptible parent CJ. 1994 to generate backcross Fj (BC,F2) populations. Unique specificities within Pea were analyzed by inoculation of these BC1F2 populations with six diagnostic crown-rust isolates. Recombination breakpoints were utilized to demonstrate that the specificities within the Pea cluster were controlled by a series of 5 tightly linked genes;

Pc81, Pc82, Pc83, Pe84, and Pc85. The Pea cluster was further targeted via bulk-segregant ampUfied-fragment-length-polymorphism (AFLP) analysis. Three markers closely linked to

Pea were identified; the Agx4 marker co-segregates with Pc85 at the end of the cluster. The sequence-tagged site (STS) markers derived from these AFLPs will be valuable for the study of the evolution of the Pea cluster and in the marker-assisted breeding of Avena.

'interdepartmental Genetics Program, ^Department of Plant Pathology, 'Com Insects and Crop Genetics Research, U.S. Department of Agriculture-Agricultural Research Service, (USDA-ARS), Iowa State University, Ames, lA 50011-1020, U.S.A. 50

Introduction

Disease resistance loci have been used in the breeding of crop plants because of their

effectiveness in preventing disease and ease of handling as single Mendelian loci. Many of

these loci are clustered in their respective genomes. The L and M loci in flax (Linum

usitatissimim) (EUis et al. 1995; Ellis et al 1999; Islam and Shepherd 1991; Pryor 1987), the

Mia locus in barley (Hordeum vulgare L,) (Giese et al. 1981; Mahadevappa et al. 1994), the

Dm3 locus in lettuce (Meyers et al. 1998; Witsenboer et al. 1995), and the Rpl locus in maize

(Zea mays) (Hulbert and Bennetzen 1991; Richter et al. 1995; Sudupak et al. 1993) are typical examples of such clustered gene organizations. Several mechanisms have been proposed to be involved in the evolution of resistance-gene clusters. Unequal recombination is one important mechanism for the generation of new resistances. For example, at the Rpl locus, evolution of new resistance specificities have been shown to be due to recombination within the locus, most likely by unequal crossing over between tandem repetitive elements

(Hu and Hulbert 1994; Richter et al. 1995). Unequal crossing over also accounts for the observed instability of /?/ji-mediated resistance (Sudupak et al. 1993; Saxena and Hooker

1968).

Several plant disease resistance genes have been cloned and molecularly characterized

(reviewed by Lawton 1997; Michelmore 1995). These genes, isolated from a variety of plant species, condition active-defense reactions against a broad range of pathogens, including bacteria, fungi, viruses, insects, and nematodes. Remarkably, most of these genes encode proteins that share conserved amino acid motifs, which suggests that certain signal events are held in common for plant defense (reviewed by Dangl 1995; Staskawicz et al. 1995).

Leucine-rich repeats (LRR) are one example of such motifs encoded by most of the plant resistance genes. LRRs are found in a variety of proteins that differ in their functions and 51 locations in the cell and are implicated in protein-protein interactions, cell adhesion, and membrane association (reviewed by Kobe and Deisenhofer 1994).

The primary function of LRRs in plant defense has been proposed to be pathogen avirulence-protein recognition (reviewed by Michelmore and Meyers 1998). Sequence comparison among the Cf-2 ICf-5 tomato leaf-mould resistance-gene families found that they possessed between 25 and 38 copies of the LRR motifs (Dixon et al 1997). This result may implicate that recombination events that vary the LRR copy number could provide a mechanism for recognition of different ligands of pathogens. Similarly, the deduced L6 and

Lll proteins differ solely in the LRR region (Ellis et al 1999). This indicates that the difference in specificity between could be determined by the LRR. Sequence comparison of different L alleles suggests that the alleles have probably involved re-assortment of accumulated point mutations by intragenic recombination and deletion (Ellis et al 1999). The detailed mechanisms by which novel specificities evolve, however, is still unknown

(Anderson et al. 1997; Thomas et al. 1997).

Puccinia coronata Corda f. sp. avenae Eriks, the causal agent of crown rust, is the most important fungal pathogen of cultivated oat (Marshall and Shaner 1992). Several crown rust resistance loci, designated Pc, have been identified and positioned in reference to molecular markers (Bush et al. 1994; Bush and Wise 1998; Jellen et al. 1995; O'Donoughue et al. 1996;

Penner et al. 1993; Rayapati et al. 1994; Rooney et al. 1994; Wise et al. 1996;). Yet, little is known about the detailed organization of these loci. The Pea locus was initially identified in a F2 population generated by crossing of the two diploid accessions, A. strigosa (C.L 3815) and A. wiestii (C.I. 1994) (Rayapati et al. 1994). The Pea locus was further characterized in a 100 Fg 9 recombinant inbred (RI) lines developed from the original Fj (Wise et al. 1996). In that study, multiple-resistance specificities to P. eoronata were postulated to be controlled by independent loci (Wise et al. 1996). In the current report, we authenticated the individual 52 genes responsible for the different specificities and determined their order within the Pea cluster. To do this, seven backcrossed F2 (BCjFz) populations between individual RI lines and the susceptible parent, C.I. 1994, were established and analyzed with diagnostic crown rust isolates. Each of the RI lines used to create the BCiFj populations possessed putative recombination breakpoints within the Pea cluster. Therefore, we reasoned that different genes that impart specificity to the diagnostic isolates could be unambiguously identified by segregation analyses. In addition, experiments were designed to identify closely linked DNA markers to the Pea resistance cluster. Three markers closely linked to Pea were identified via AFLP-based bulk-segregant analysis. One of the markers, Agx4, co-segregates with

Pe85, the gene that provides resistance-specificity to crown-rust isolate 202.

Results

DISSECTION OF OAT CROWN-RUST RESISTANCE GENES WITHIN THEFCA CLUSTER

In our early studies, crossovers among resistance-specificities within the Pea cluster were postulated by recombination analysis of 100 recombinant inbred (RI) lines inoculated with 11 isolates of P. eoronata (Wise et al. 1996). To conclusively demonstrate the positions of the different specificities within the Pea cluster, seven RI lines with putative recombination breakpoints within the cluster were backcrossed to the susceptible parent, C.I. 1994. The seven backcrossed hybrids were selfed to generate large BCiFj populations. Plants from the seven BCiFj populations were inoculated individually with six diagnostic crown rust isolates and readings were taken 7 days post inoculation. For each isolate-BCiFj combination, the infection type reaction (ITR) should segregate 3:1 (resistant :susceptible) if the original RI lines used in the backcross possessed a resistance specificity to the isolate used for the inoculation. If, however, the RI lines did not contain the resistance specificity to the isolate, the BC1F2 population would be entirely susceptible. To test these assumptions, all forty-two 53

combinations of BCiFj populations and the 6 diagnostic isolates were analyzed. At least 16

plants were used for each test to ensure a 99% probability of observing a susceptible plant if

a single dominant gene controls the specificity.

Analysis of the A8060 x C.L 1994 BCiFj indicates the presence of specificities to all six

isolates in A8060

The results of inoculation of six isolates on the A8060 x C.I. 1994 BCiFj population are listed in Table l.A. Segregation of infection type was observed in the.BCiF2 population in response to infection by all six isolates. The results of chi-square analyses were consistent with a ratio of 3:1 (resistant to susceptible) within each BC1F2 population in response to each of the six isolates. The individual genetic factors that determine the unique specificities at the Pea locus, therefore, could not be differentiated in the A8060 x C.I. 1994 BC1F2 population.

Analysis of the A8028 x C.1.1994 BC1F2 indicates that an independent gene controls the

R202 resistance specificity to isolate 202

The results of the multiple inoculation analysis on the A8028 x C.I. 1994 BC1F2 population are listed in Table l.B. Segregation occurred in response to the infection by

PC62, 276, 263, and 290, and PC54. All of these segregation ratios were in good fit to 3:1

(resistant to susceptible), indicating inheritance of dominant specificities in response to each of these isolates. In contrast, all nineteen plants of the BCjFj population and the RI A8028 were susceptible to the infection by isolate 202, indicating the absence of its corresponding resistance specificity. Hence, A8028 contains a recombination breakpoint between the locus determining specificity to 202 and the loci encoding specificities to the other isolates. This result indicates that a unique gene controls specificity to isolate 202. Table 1. Frequencies of phenotypic classes and chi-square goodness-of-fit tests for infection type reactions (ITR) to 6 isolates of P. coronata on BCiFj progeny of diploid Avewfl

Infection type reaction"

BC1F2 Individuals Presence of resistance BC.F, specificity Cross Isolate RI line' Resistant Susceptible value (3:1) Probability' 1 2 3 4 Total

A:A8060 PC54 2 1 16 6 23 0.01 0.95-0.90 -h" X C.I. 1994 290 2 15 6 21 0.14 0.90-0.70 + 276 2 3 14 8 25 0.65 0.50-0.30 + 263 2 16 9 1 6 32 0.167 0.70-0.50 + PC62 1 4 10 6 20 0.267 0.70-0.50 + 202 2 8 14 3 4 29 1.94 0.20-0.10 +

B:A8028 PC54 2 13 7 7 27 0.01 0.95-0.90 + xC.1.1994 290 1 18 4 22 0.55 0.50-0.30 + 276 2 19 4 5 28 1.712 0.20-0.10 + 263 2 26 10 1 9 36 0.26 0.75-0.70 + PC62 1 10 15 1 8 34 0.012 0.95-0.90 + 202 4 19 19 57 <0.001* + 2 10 13 2 6 31 0.017 0.95-0.90 + 290 1 21 4 1 5 32 0.06 0.90-0.70 +

276 2 2 32 11 2 47 0.177 0.70-0.50 -1- 263 2 21 7 28 0.00 >0.95 + PC62 3 24 6 30 90 <0.001* 202 3 15 7 22 66 <0.001* PC54 1 17 8 25 065 0.50-0.30 + 290 2 4 12 5 3 27 0.89 0.50-0.30 +

276 2 4 13 2 8 27 2.09 0.20-0.10 + 263 1 17 10 27 2.09 0.20-0.10 + PC62 4 25 25 75 <0.001* 202 3 16 9 25 75 <0.001* - PC54 _ _ _ _ _ 0.019 0.95-0.90 + 290 2 6 5 3 1 15 0.02 0.90-0.70 +

276 3 11 1 3 15 0.02 0.90-0.70 + 263 4 19 19 57 <0.001* PC62 4 37 37 111 <0.001* 202 3 5 10 15 45 <0.001* Table 1. (continued)

F:A8085 PC54 2 13 1 5 19 0.44 0.90-0.70 +

X C.1.1994 290 4 29 29 87 <0.001* -

276 3 18 14 32 96 <0.001* -

263 4 26 26 78 <0.001* -

PC62 3 30 30 90 <0.001* -

202 3 22 4 26 78 <0.001* -

G:A8098 PC54 2 1 13 8 22 1.25 0.30-0.20 + xC.1.1994 290 2 19 3 22 66 <0.001* -

276 3 3 24 27 81 <0.001* -

263 4 27 27 81 <0.001* -

PC62 4 30 30 90 <0.001* -

202 3 16 16 48 <0.001* - ' Three seedlings were evaluated of each parental RI line. '' Infection type reaction (ITR) of 0,1, and 2 are designated resistant. ITR of 3 and 4 are designated susceptible. ' An asterisk indicates the probability of not detecting a significant difference from a 3:1 ratio (resistant to susceptible) in the BC,F2 population. •' A "+" indicates the presence of the resistance specificity while a indicates the absence of the resistance specificity. Parental line C.1.1994 was completely susceptible to all six crown-rust isolates. 57

Analysis of the A8073 x C.I. 1994 and A8076 x C.I. 1994 BCiFj populations indicates

that the R62 resistance specificity is controlled by an independent gene that confers

resistance to isolate PC62

The results of inoculation of the A8073 x C.I. 1994 BCiFjpopulation by six crown-rust

isolates are shown in Table l.C. The parental line A8073 displayed a moderate level of

resistance to isolate PC54, 276, 290, and 263. The observed 3:1 segregation ratios in

response to these isolates indicated the presence of single dominant loci conferring these specificities. In contrast, RI A8073 and its derived BC1F2 population were entirely susceptible to isolates PC62 and 202. Therefore, the A8073 line possesses a recombination breakpoint between the loci encoding specificities to isolates 202 and PC62 and the specificities to the other four isolates. Because the specificity R202 was controlled by an independent gene and separated from the specificity to PC62 (R62) in the A8028 line (Table

l.B), the gene controlling specificity to PC62, therefore, is independent from the gene controlling specificity to isolate 202. The same result was demonstrated in the segregation analysis of the infection type reactions on the A8076 x C.I. 1994 BCiFj population (Table l.D). In this case, segregation of FTR was observed in the BCiFj population to the infection by 276, 290, 263, and PC54, but not to PC62, or 202. Chi-square goodness-of-fit tests indicated that presence of single dominant specificities to 276,290,263, and PC54, but not to

PC62 and 202. Thus, recombination between the two loci that confer specificity to isolate

202 and PC62 indicates again that there exists a unique gene for specificity to isolate PC62.

Analysis of the A8096 x C.I. 1994 BCiF^ indicates that a unique gene controls the specificity to isolate 263

Table l.E shows the segregation data of infection type reactions of BC1F2 population derived from the cross of the RI A8096 and C. I. 1994. The ratios of the resistant to susceptible plants were in good fit to 3:1 to the infection by isolate PC54, 296, and 290, 58 indicating the presence of single dominant specificities to these isolates. In contrast, both the

RI A8096 and its derived BC1F2 population were susceptible to the infection by the other three isolates: 263, PC62, and 202. As indicated above, specificities to 202 and PC62 were controlled by two separate genes and can be separated from specificity to 263 (Table l.D).

Therefore, there is a single dominant gene that determines specificity to isolate 263 and it is separate from the genes that determine the specificities to isolates 202 and PC62.

Analysis of the A8085 x C.I. 1994 and A8098 x C.I. 1994 BC^Fj populations indicates that there is a unique gene that controls specificity to isolate PC54

The segregation data of infection type reactions in A8085 x C.I. 1994 BCjFj population are shown in Table l.F. Both RI A8085 and its BC,F2 population were susceptible to the infection by five isolates: 276,290, 263, PC62 and 202. In contrast, RI A8085 was resistant to the sixth isolate, PC54. Among the twenty-five individual plants in the BCjFj population,

19 displayed a resistant phenotype and 6 were susceptible, consistent with 3:1 ratio, indicating the presence of a single dominant gene. RI A8085, therefore, displayed a distinct recombination breakpoint between a locus that determines specificity to isolate PC54 and specificity to the other 5 isolates. This result was consistent with the results of the inoculation of the A8098 x C.I. 1994 BQFz population. Table l.G shows the segregation data of infection type reactions in this population. Like RI A8085 and its derived BC1F2 population, RI A8098 and all member plants in its BC1F2 population were susceptible to 276,

290, 263, PC62 and 202. Therefore, a single dominant gene was shown to control the specificity to isolate PC54.

The specificity to isolates 276 and 290 can not be further differentiated by recombination analysis

A recombination breakpoint has not yet been identified between the specificity to 276 and

290 in the seven RI lines. RI lines A8060, A8028, A8073, A8076, and A8096 were resistant 59 to both isolates. Chi-square analyses indicated that single dominant genes controlled the two specificities (R276/R290) in those RI lines. At the sanne time, RI lines A8085, and A8098 and their derived BCiFj populations did not display resistance to either isolate. This opens the possibility that either a single gene or two independent, but tightly linked genes controlled the two specificities. However, we could not determine which case was true due to lack of recombination.

Table 2. Segregation of the infection type reactions in 42 isolateiBCjFj population combinations.

BC1F2 population Isolate R-gene A8060 A8028 A8073 A8076 A8096 A8085 A8098 PC54 Pc81 17R:6S 20R:7S 23R:8S 17R:8S 12R:5S 13R:6S 14R:8S 290 Pc82 15R:6S 18R:4S 25R:6S 16R:8S 11R:4S 0R:29S' 0Rr:i2S 276 Pc82 17R:8S 19R:9S 34R;13S 17R:10S 11R:4S OR:32S 0Rr27S 263 Pc83 25R:7S 36R:10S 21R:7S 17R:10S i 0R:I9S 0R:26S 0R!30S PC62 Pc84 14R:6S 25R;9S i 0R30S 0R;25S OR:37S 0R:30S OR:30S 202 Pc85 22R:4S 0R;19S 0R:22S 0R:25S 0R:}5S 0R:26S OR:l«iS

" The shaded areas represent isolate: BC1F2 population combinations in which there are no segregation of the infection type reactions while non-shaded areas represent isolate: BCiFj population combinations with the inheriance of single dominant resistance genes. The interface represents the locations of recombination breakpoints on the seven RI lines.

The arrangement of resistance genes is determined by the distribution of recombination breakpoints among the resistance specificities within the Pea cluster

As demonstrated above, five independent genes were separated within the Pea locus by recombination analysis. The order of these resistance genes in the Pea cluster was determined by the distribution of the recombination breakpoints in the RI lines. Table 2 summarizes the segregation data of infection type reactions of all the isolates and BC1F2 population combinations. The first recombination breakpoint occurred between a locus 60

conferring resistance specificity R54 and the locus conferring specificity R290/R276 in RI

lines A8085 and A8098. The second recombination breakpoint occurred between loci

conferring resistance specificities R290/R276 and R263 in RI A8096. The third

recombination breakpoint occurred between loci conferring resistance specificities R263 and

R62 in RI lines A8073 and A8076. The fourth recombination breakpoint was positioned

between loci conferring resistance specificities R62 and R202 in the RI A8028. Taken

together, the five specificities in the Pea cluster were arranged as R54-R290/R276-R263-

R62-R202 and the gene designations given are Pc81-Pc82-Pc83-Pc84-Pc85 (Fig. 3).

BULK-SEGREGANT AFLP ANALYSIS IS USED TO TARGET THE RESISTANCE GENES IN THE

PcA CLUSTER

Tightly linked DNA markers have three important roles in the study and utilization of plant disease resistance genes. First, they are valuable in the marker-assisted disease- resistance breeding. Second, flanking markers can be used to determine the molecular and genetic events that are involved in the evolution of resistance specificities (Islam and

Shepherd 1991; Lawrence et al. 1995; Sudupak et al. 1993). Finally, DNA markers that are tightly linked to the target genes are utilized to provide entry points for map-based gene cloning (Lagudah et al. 1997; Tanksley et al. 1995). Previously, only two loosely linked

DNA markers, Xisu2192 and Operon C18, were found to flank the Pea cluster (Rayapati et al. 1994; Wise et al. 1996; Yu et al. 1996). Therefore, we used bulk-segregant AFLP analysis to target this resistance gene-rich region. The flanking DNA markers Xisu2192 and

Operon C18 were used to delineate the region linked to Pea. Utilizing this information, two

DNA bulks were constructed to enrich for candidate markers that are located within a specified interval surrounding the Pea cluster. Bulk A consisted of DNAs of sixteen RI lines resistant to isolate 202. In half of these individuals (RI lines 8003, 8016, 8031, 8034, 8037,

8063, 8078, and 8090), crossovers occurred between the Pea cluster and flanking marker. 61

Xisu2192, but there are no crossovers between the Pea locus and the opposite flanking

marker, Operon C18. In the other half of the plants (RI lines 8033, 8049, 8061, 8070, 8071,

8084, 8087, and 8093), there are no crossovers between the Pea cluster and Xisu2192, but

crossovers occurred between the Pea cluster and Operon CI 8. The same principle was

utilized to construct bulk B except that plants in this group are disease-susceptible. Among

these plants, eight (RI lines 8007, 8010, 8023, 8026, 8035, 8048, 8050, and 8057) have

crossovers between the Pea cluster and Xisu2192 while the other eight (RI lines 8008, 8009,

8030, 8038, 8056, 8068, 8094, and 8097) have crossovers between the Pea cluster and

Operon CIS. Therefore, both bulks have homozygous genotypes for the resistance-related

region and heterozygous genotypes elsewhere.

Three AFLP markers closely linked to the Pea cluster are identified; Agx4 co-segregates

with Pc85.

Two hundred and fifty-six EcoRI and Msel AFLP primer pairs were used to amplify DNA on the diploid oat parents and the designated bulks. Of 1,475 fragments that were polymorphic between the parents, 15 were found to be potential DNA markers linked to Pea.

The AFLP primer pairs that were used to amplify these candidate markers were tested on the complete A. strigosa (C.I. 3815) x A. wiestii (C.I. 1994) recombinant inbred population.

Three of the original 15 AFLP markers were closely linked to the Pea resistance-gene cluster. The marker amplified with AFLP primer pairs E-ACA and M-CAA detected a susceptible (C.I. 1994)-specific DNA fragment. This marker was designated Agx4, corresponding to the fourth polymorphic band numbered from the top of the sequence gel.

Fig. 1 illustrates the segregation of the amplified fragment on the population. Among 99 RI lines, 50 that displayed a susceptible phenotype with isolate 202 also showed strong amplification with the primers, E-ACA and M-CAA. In contrast, the other 49 lines that displayed a resistant phenotype did not amplify with this primer set, indicating that this Cr C^ Resistant Rl lines Susceptible Rl lines

Fig. 1. Segregation of the amplified fragment on the A. strigosa (C.I. 3815) x A. wiestii (C.I. 1994) recom binant inbred population obtained with AFLP primer pair E-AAC and M-CAA. The amplified fragment is approximately 450-bp and the fourth polymorphic fragment numbered from the top of the sequence gel. The first two lanes show the amplification polymorphism on the two parents at the positionindicated by the arrow. ^ eS*

^ Resistant Rl lines Susceptible Rl lines

600bp Agx4-a Agx4-b Agx4-c lOObp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Fig. 2. Segregation of the amplified fragment on the A. strigosa (C.I.3815) x A. wiestii (C.I. 1994) recombinant inbred population obtained with Agx4 specific primer pair of ACA-IOF (5'-ACCAATTCACAGACCGAG-3') and ACA-293R (5'-CCAACGCAATCAAAAGTCC-3'). The first lane is 100-bp DNA ladder. The next two lanes show the sequence-tagged specific amplification polymorphism on the two parents. The last 16 lanes show the segregation pattern of the polymorphism. 64

(cM) E11M10-3a 0.6 E13M11-13a E5M6-8a

10.2

Xisu2192

20.4 Agx7

Pc81 4.3 PC82-276 PC82-290

0.6 Pc83 0.5 Pc84 3.8 Pc85 Agx4

11.3 Agx9

Operon C18

Fig. 3. Linkage map of the resistance genes in the Pea cluster and flanking markers. Pc81, Pc82, Pc83, Pc84, Pc85 were resistance genes identified in the Pea cluster, which correspond to six resistance specificities, R54, R270yR290, R263, R62, and R202 respectively. Agx4 Agx9 are two AFLP derived STS markers. Agx7, ElM10-3a, E13Mll-13a, and E5Mll-8a are AFLP markers. 65

marker co-segregated with the resistance gene, Pc85 (Fig. 2). The AFLP fragments

corresponding to Agx4 were cloned, sequenced, and i4gj:4-locus specific PCR-primer pairs

were developed. Fig. 3 shows a subset of the segregation data between the resistance gene,

Pc85 and the amplified DNA fragments specific to the Agx4 specific primer pairs ACA-IOF

and ACA-293R (see Materials and Methods). Three DNA firagments of different lengths

(Agx4-a, Agx4-b, and Agx-4c) were detected in the susceptible RI lines. All of them show

co-segregation with Pc85 although the level of amplification was variable. Among the three

DNA fragments, only Agx4-c had the expected DNA length (250-bp) defined by the two

designed primers. The other two fragments, Agx4-a (570-bp) and Agx4-h (480-bp), were

much larger, suggesting that the primer sequences were repeated in this region. This

repetitiveness was further demonstrated by Southern hybridization. When the Agx4

amplified DNA fragment was used as a hybridization probe on restriction-digested oat

genomic DNA, a uniform smear was revealed both on the resistant and the susceptible

parents (data not shown).

Two other AFLP markers, designated Agx9 and Agx7, were identified by AFLP primer

pair E-ACC and M-CAG and AFLP primer pair E-AAA and M-CGT, respectively. In

contrast to the A^x^-specific AFLP primer pair, which detected a susceptible-specific

marker, the Agx9-specific AFLP primer pair only amplified a fragment from DNA of the

resistant parent. Agx9 is positioned between the resistance gene Pc85 and the Pea-flanking

marker OperonC18. The PGR primer pairs, AGG24F (5'-GAATGTTGTGGTGAAAGTG-

3') and AGG117R (5'-AACAGGTCTGACGAGGGAA-3') were developed for Agx9 and

mapped at the same locus as Agx9. Agx7, like Agx4, is susceptible specific and positioned

between resistance gene Pc81 and flanking marker Xisu2192. PGR primer pairs for Agx7

were also developed. However, due to lack of polymorphism among the amplified fragments, a definite map position was not possible to determine. The map positions of these 66 two markers and the five crown rust resistance genes within the Pea cluster are also illustrated in Fig. 2.

The Agx4-derived PCR primer pairs can be used to follow segregation of the resistance alleles

One of the immediate applications of cosegregating markers is that they can be used to follow DNA segments linked to or containing the resistance loci during plant breeding. We tested the Agx-4 specific primer pair ACA-43F and ACA-293R on a BC1F2 population derived from the cross A8060 x C.L 1994. ACA-43F is the second Agx-4 specific forward- primer tested on the RI population after ACA-IOF and gave an identical result to ACA-IOF when paired with ACA-293R. When inoculated with isolate 202, a ratio of 3:1 (resistant: susceptible) was detected, indicating an inheritance of a single dominant gene (Table l.A).

Agx4 primers amplify a fragment from C.I. 1994 that cosegregates with Pc85. Fig. 4 illustrates the segregation of the Agx4 amplified fragment from resistant and susceptible

BC1F2 plants. The 250-bp Agx4 locus-specific fragment was amplified from all 5 susceptible plants, illustrating that this fragment cosegregates with Pc85. By utilizing this marker, we can identify plants homozygous (plants in lane 13,14, 15,16 and 17) or segregating (plants in lane 4, 5, 7, 8 and 9) for the susceptible allele. Thus, the Agx4 DNA marker can be used in recombination analysis of the Pea cluster and also to select against the susceptible allele during plant breeding.

Discussion

Crown rust, caused by the infection of fungal pathogen Puccinia eoronata, is one of the most damaging fungal diseases on cultivated oats (Marshall and Shaner 1992). Crown rust can greatly reduce grain yield and quality. The most effective way to fight the disease is by breeding resistant plants (Marshall and Shaner 1992). Resistances used in oat breeding are 67

mosdy derived from single genes. The main problem of utilizing the single-gene-controlled

resistance is that virulence patterns of rust populations can shift dramatically when a single

gene for resistance is widely distributed (Harder and Haber, 1992). Therefore, single-gene

resistances can be rapidly overcome (Browning, 1973; Frey, 1992).

The Pea resistance cluster contains several loci that confer distinct specificities to the crown rust pathogen (Wise et al. 1996). Five unique resistance genes within the Pea cluster

have been differentiated by this study. The combined diversity of the unique resistance specificities could be important to breeding for crown-rust resistance. All resistance genes within the Pea cluster are present in coupling in the resistance parent C.I. 3815. This observation opens the possibility that genes within the cluster could be utilized together if the whole gene cluster could be transferred into commonly used cultivated varieties.

In the original recombinant-inbred population, resistance-related genes that were contained within the C.I. 3815 resistant parent may be well segregated, leading to formation of individual RI lines that have diverse genetic backgrounds. It may lose one or more of those resistance-related genes and therefore, only confer a compromised resistance to a specific isolate even if an individual RI possesses the corresponding resistance gene. The infection types of these RI lines are vulnerable to the change of environment and difficult to assay. Therefore, to authenticate the genes responsible for these unique resistance specificities, it was necessary to determine the position of recombination breakpoints among the genes. In this study, BCiFj populations were used to determine recombination breakpoints among the resistance specificities within the Pea cluster. The segregating BCiFj populations have an advantage over single RI lines for identification of recombination breakpoints among resistance specificities. This is because the infection types of a RI can be unequivocally determined in the BCjFj populations. Response to crown-rust isolates would fit a 3:1 ratio if the RI has a resistance specificity to the isolate used for inoculation. For Resistant Fz lines Susceptible Fz lines

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fig. 4. Segregation of the amplified fragments on the A8060 x C.I. 1994 BCiFz population obtained with Agx4 specific primer pair of ACA-43F (5'-AGTACCCGTTTCAGAGGAG-3') and ACA-293R (5'-CCAACGCAATCAAAAGTCC-3'). The first lane is the 100-bp DNA ladder. The second lane and the third lane show the sequence-tagged specific amplification polymorphism on the two parents at position of 250-bp (pointed by arrow). The next 9 lanes (from lane 4 to lane 12) show the segregation of this susceptible-specific fragment among the resistant plants in which half of the plants are heterozygous. The last 5 lanes (from lane 13 to lane 17) show its segregation among 5 susceptible F2 plants. 69 example, the RIA9060 is partially resistant to five crown-rust isolates, PC54, 290, 276, 263 and 202. However, the ratios of the resistant to the susceptible plants fit a single-dominant- resistance-gene-inheritance in the A9060 x C. 1.1994 BC1F2 population.

The challenge to plant-breeding is to provide durable resistance to rapidly evolving pathogens. Resistance-gene clusters will be very important in that they are a rich source of genetic diversity (Meyers et al. 1998; Richter et al. 1995; Sudupak et al. 1993; Witsenboer et al. 1995). New resistance specificities can be generated within these clusters by gene conversion, duplication, and unequal crossovers (Hu and Hulbert 1994; Richter et al. 1995).

Thus, the identification of five unique genes with the Pea cluster provides a foundation on which these unique genes can be further characterized by recombination analysis. Utilization of the Pea resistance gene cluster in an oat resistant breeding program would lead to a rich source of resistance specificities to crown rust isolates. A chromosomal segment carrying the

Pea cluster could be transferred into cultivated hexaploid oat germplasm by means of conventional backcossing program (Sadanaga and Simons 1960). This could result in durable resistance to the crown-rust disease, solving problems related to the utilization of single resistance genes. With our current data, the Pea cluster becomes a more amenable target for plant breeding.

Bulk-segregant analysis and AFLP are a highly efficient combination to target the resistance genes in the Pea cluster

Identifying molecular markers tightly linked to the Pea cluster opens a number of opportunities for both basic and applied applications. One of the immediate effects is using those markers for the study of evolution of the resistance genes within the gene cluster by observing the position change of the flanking markers relative to individual genes during the genetic recombination process. This strategy has been successfully used to find the genetic mechanisms for instability of /?p7-mediated resistance (Sudupak et al. 1993; Saxena and 70

Hooker 1968), and for generation of new resistance specificities (Hu and Hulbert 1994).

Bulk-segregant- and AFLP-analysis has been a highly efficient combination in gene targeting

(Michelmore et al. 1991; Becker et al. 1995; Thomas et al. 1995; Biischges et al. 1997).

Over twenty thousand amplified DNA fragments were screened in this study. Of these, three markers were found to cover the Pea resistance-gene region. These three markers are currently the only DNA markers that are closely linked to the Pea resistance cluster among over five hundred DNA markers on the A. strigosa (C.I. 3815) x A. wiestii (C.I. 1994) recombinant inbred population (G. X. Yu and R. P. Wise, unpublished).

Molecular markers have been successfully used in map-based gene cloning and marker- assisted breeding (reviewed by Tanksley et al. 1995). The Agx4 locus-specific amplified fragment cosegregated with the susceptible plants in the A8060 x C. I. 1994 BC1F2 population, suggesting that this marker can be used to identify individual plants that carry the target resistance locus, either heterozygous or homozygous. It can be predicted that this primer pair will play an important role in the recombinational analysis within the Pea cluster and in the marker-assisted breeding of Avena.

Materials and Methods

Mapping populations

The diploid i4ve/ja population was generated by crossing A. strigosa (C.I. 3815) with A. wiestii (C.I. 1994) (Rayapati et al. 1994). A. strigosa is resistant to 40 isolates of P. coronata in the ISU collection whereas A. wiestii is susceptible to the same isolates (Wise and Gobelman-Wemer 1993). One hundred F^.^ RI lines generated by single seed descent from an original Fj mapping population were kindly provided by Dr. M. Lee (Iowa State

University). To differentiate genetic factors that control multiple specificities in the Pea locus, seven RI lines that contained putative recombination breakpoints within the Pea 71 cluster were backcossed to the susceptible parent C.I. 1994. The seven backcrossed hybrids were selfed to generate BCiFj populations.

Rust inoculations

To authenticate the individual genes responsible for the different specificities and determine their order within the Pea cluster (Rayapati et al. 1994), 6 oat crown-rust isolates were used to inoculate over sixteen individuals from each of the BCjFj populations described above. Crown-mst inoculation and incubation were processed under our standard conditions

(Bush et al. 1994). Disease development was assessed at 7 days after inoculation. On a 0 to

4 scale using the method of Murphy (1935), infection type reactions (ITR) of 0 or 1 are considered resistant and 3 or 4 are considered susceptible.

Table 3. EcdRi and Msel AFLP primers represented by their distinct selective base pairs for selective AFLP amplification.

Source Primer Gibco-BRL Iowa State DNA Sequencing and Synthesis Life Technologies, Inc Facility (Ames, lA) EcdSl AAC, AAG.ACA, ACC, AAA, AAT, AGA, AGT, ACG, ACT, AGC, AGG ATA, ATC.ATG, ATT Msel CAA,CAC,CAG,CAT, CCA, CCC, CCG, CCT, CTA, CTC, CTG, CTT CCA, CGC, CGG, CGT ' The three-letter sequence designates the selective bases that were added to the 3' end of the core sequences. The core sequence of £coRI primers is 5'-AGACTGCGTACCAATTC-3' and the core sequence of Msel primers is 5'-GATGAGTCCTGAGTAAC-3'. The first nucleotide A in the three-letter sequence of the £coRI primer and the first nucleotide C in the three-letter sequence of Msel primer were used as selective bases in the pre-selective amplification. 72

AFLP protocol

The initial primers used in the AFLP-gene targeting were from the large genome kit

supplied by GIBCO/BRL Life Technologies, Inc. (Gaithersburg, MD). Supplementary

primers were designed by Fusheng Wei of the Wise lab and were synthesized at the Iowa

State DNA Sequencing and Synthesis Facility (Ames, lA). The primers and their sequences

are shown in Table 3.

To prepare the AFLP template, DNA from each individual RI was digested

simultaneously with the restriction endonucleases, EcoRI and Msel. Subsequently, the

genomic DNA fragments were ligated to both EcoRl and Msel adapters, generating DNA

templates for PGR amplification. Amplifications were performed in two consecutive steps.

The first step selectively amplified the adapter-ligated DNA fragments with one-selective-

base primers, nucleotide A for the EcoRl primer and C for the Msel primer. In the second

step, the pre-selectively amplified products were diluted and amplified by the EcoRl and

Msel primer pairs with three-selective-bases. In this step, the £coRI primers were labeled

with [^^PJ-y-ATP. The selective amplified PGR products were mixed with one volume (20

|il) formamide dye: 98% formamide, 10 mM NajEDTA, 0.005% each of xylene cyanol and

bromophenol blue and denatured for 4 minutes at 90° G. The samples (2.4 |il each) were size

fractionated through a 7% denaturing polyacrylamide gel (Long Ranger™, FMC, Rockland,

Maine) in 1 x TBE buffer at 65W for 2 hours. The gel was wrapped with plastic wrap and

exposed to BIOMAX Kodak Scientific Imaging film at -80° G for approximately two days.

Cloning amplified DNA Fragments from sequence gel

The bands corresponding to the desired amplified DNA fragments were cut out of sequence gel and diluted in 10 |Xl of ddH20 overnight at 4°G. The DNA were first amplified

with 2 |J,1 of the diluted solution combined with single EcoRI AFLP primer originally used for ten cycles to enrich the target sequence. Subsequently, 2 (xl of the PGR product was 73 amplified again with both die £coRI and Msel primers. Amplified PGR products were checked in the mini-agarose gel and cloned into pGEM-T easy vector (Promega, Madison,

WI). DNA fragments from single clones were verified by comigration with the original

AFLP reaction. Confirmed clones were sequenced in the Iowa State DNA Sequencing and

Synthesis Facility (Ames, lA). Specific primers were developed from those sequences using computer program OLIGO 5 (Molecular Biology Insights, Inc. USA). Agx4 and Agx9 are two AFLP markers identified in this research. They are closely linked to the Pea resistance cluster.

Bulk design for targeting the Pea cluster

To find DNA markers tightly linked to the Pea cluster, two bulks were constructed (see the detailed explanation in Results). Sixteen disease-resistant and sixteen disease-susceptible

RI lines were selected for each pool. Sixteen lines were chosen so that that probability that unlinked markers were represented in each pool was greater than 0.99 to reduce the frequency of false-positive signals. The RI lines in the pools were treated individually in die

AFLP processes as described above until selective amplification. Subsequentiy, they were pooled together at a ratio of one to one.

Data analysis

Data were entered into MapManager V2.6.5 (Manly 1993) and further prepared for

MapMaker by using the 'Export for MapMaker' command prior to mapping in MapMaker 3.0

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Genome 39:155-164. 82

CHAPTER 4. AN ANCHORED AFLP AND RETROTRANSPOSON-BASED

MAP OF DIPLOID 4

A paper to be submitted to Genome

Gong-Xin Yu''^, and Roger P. Wise^'^'^

Abstract

Based on a recombinant inbred (RI) population developed from a cross between A.

strigosa and A. wiestii, a saturated genetic map of diploid oat was developed. This map

includes 372 AFLP and 78 S-SAP markers, six crown-rust resistance genes, a cluster of

resistance-gene analogs, one morphological marker, and is anchored by 45 grass-genome

RFLP markers. This new A. strigosa x A. wiestii RI map is colinear with a diploid Avena

map from an A. atlantica x A. hirtula F2 population. However, some linkage blocks were

rearranged as compared to the RFLP map derived from the progenitor A. strigosa x A. wiestii

Fj population. Clustering of markers led to formation of highly saturated linkage blocks.

Mapping of Bare-1-like sequences by sequence-specific AFLP indicated that related

retrotransposons had considerable heterogeneity and widespread distribution in the diploid

Avena genome. Novel amplified fragments detected in the RI population suggested that

some of these retrotransposon-like sequences may be active in diploid Avena. This

framework map will be useful in gene cloning, genetic mapping of qualitative genes, and

positioning QTL of agricultural importance.

'Interdepartmental Genetics Program, ^Department of Plant Pathology, ^Com Insects and Crop Genetics Research^ U.S. Department of Agriculture-Agricultural Research Service, (USDA-ARS), Iowa Slate University, Ames, lA 50011-1020, U.S.A. 83

Introduction

Molecular and genetic linkage maps have been successfully applied to various endeavors such as gene targeting and positional cloning (Tanksley et al. 1995). Several kinds of molecular markers have been utilized in establishing these linkage maps in plants and animals. They differ in principles, applications, polymorphism rates, and cost and time requirements. Restriction fragment length polymorphisms (RFLPs) is one of those classes of markers commonly used since proposed by Botstein et al. (1980). In addition to their ability to detect homologous DNA sequences in the genome, RFLPs also detect heterologous sequences in the genomes of different species. Therefore, they provide an opportunity to address important biological questions about the evolution of plant genomes (Ahn et al.

1993a; Ahn and Tanksley 1993; Bennetzen and Freeling 1993; Yu et al. 1996). Since the development of polymerase chain reaction (PGR) technology (Saiki et al. 1988), several

PCR-based mapping techniques have been developed. These techniques are well adapted to efficient, non-radioactive DNA fingerprinting of different genotypes. These include cleaved amplification polymorphisms (CAPs) (Konieczny and Ausubel 1993), simple sequence repeats (SSRs) (Tautz 1988), and randomly amplified polymorphic DNA (RAPDs) (Williams et al. 1990). The polymorphism information displayed by RFLPs and these PGR based techniques, however, is limited. This restrains us from quickly targeting specific genes and saturating existing maps. The rapid detection of polymorphism is very important in gene discovery by positional cloning, especially in plants with complex genomes.

Amplification fragment length polymorphisms (AFLPs) and retrotransposon-based sequence specific amplification polymorphisms (S-SAPs) are both powerful techniques for

DNA fingerprinting. The usefulness of the AFLP method (Zabeau and Vos 1993) has been amply demonstrated (Becker et al. 1995; Thomas et al. 1995; Vos et al. 1995; Biischges et al.

1997). AFLP combines the strengths of sensitive PGR amplification and the high resolution 84

of polyacrylamide gel electrophoresis. The main advantage of this technique is that it is

possible to detect single nucleotide changes and differentiate over one hundred amplified

DNA fragments in a single experiment in the absence of prior sequence knowledge.

Retrotransposons are the most common class of eukaryotic transposable elements.

Retrotransposons include long terminal repeat (LTR) and non-LTR classes (Boeke and

Corces 1989; Smyth, 1993; Kumar 1996). The most studied group of LTR retrotransposons

is the Tyl-copia group. The Tyl-copia class of retrotransposons exhibits widespread

distribution and heterogeneity in plant genomes (Flavell et al. 1992; Voytas et al. 1992;

Lindauer et al. 1993; Kumar 1996; Waugh et al. 1997). To utilize this widespread

heterogeneity, retrotransposon-based S-SAP was designed based on the novel properties of

the retrotransposons and the AFLP technique. A retrotransposon-sequence-specific primer was designed from a highly conserved LTR sequence of a Bare-1-like retrotransposon, a Tyl- copia like element present in 70,000 copies in the barley genome (Waugh et al. 1997). When paired with random AFLP primers, additional polymorphisms due to retrotransposon-specific variations can be detected. In the barley genome, a 25% increase in the rate of polymorphism has been observed with retrotransposon-based S-SAP as compared to standard

AFLP (Kumar 1996; Waugh et al. 1997).

Three low-resolution genetic maps have been constructed for Avena based on RFLP, two for diploid (O'Donoughue et al. 1992; Rayapati et al. 1994a; Van Deynze et al. 1995b) and one for hexaploid Avena (O'Donoughue et al. 1995). These maps provide a foundation on which to pursue more targeted gene-discovery efforts. Thus, our objective here was to survey the distribution of both amplified fragment length polymorphisms (AFLPs) and Bare-

1-like retrotransposon sequence-specific amplification polymorphisms (S-SAPs) in diploid

Avena genome, and to establish a saturated map for the A. strigosa x A. wiestii RI population.

Anchor probes were used to assign the AFLP and S-SAP markers to known linkage groups. 85

A total of five hundred and nine markers, including three hundred and seventy two AFLP markers and seventy eight S-SAP markers were mapped in seven linkage groups. These data will be uploaded to the USDA-Graingenes database, creating a framework for public utilization.

Materials and Methods

Mapping population

The diploid Avena population was generated by crossing A. strigosa (C.I. 3815) with A. wiestii (C.I. 1994) (Rayapati et al. 1994a). Crown rust, caused by Puccinia coronata f. sp. avenae, is an important fungal disease of cultivated oat. A. strigosa is resistant to 40 isolates of P. coronata in the ISU collection whereas A. wiestii is susceptible to the same isolates

(Wise and Gobelman-Werner 1993). One hundred Fg.^ RI lines generated by single seed descent from the original Fj mapping population were kindly provided by Dr. M. Lee (Iowa

State University).

DNA extraction, Southern blotting, and hybridization

Fresh leaf tissue samples were harvested from greenhouse-grown plants and frozen at -

80OC. DNA isolation and RFLP analysis were performed as described previously (Bush et al. 1994; Wise and Schnable 1994). Five enzymes. Oral, EcoRI, EcoRV, HindEl, and Kpnl were used to restrict oat genomic DNA for polymorphism surveys. The stringency of post- hybridization washes was modified because of the lower Tm associated with some of the heterologous DNA probes. The membranes were processed 4 x 30 minutes at 65°C in 1 x

SSPE (20X SSPE, 3.6 M NaCl, 0.2 M NaH2P04,20 mM NazEDTA, pH 7.4), 0.1 % SDS and subsequently exposed to Kodak X-Omat AR film for 3-5 days at -80°C with one or two intensifying screens. 86

Table 1. AFLP primers used for parental surveys and genetic mapping in diploid Avena £coRI core sequence Msel core sequence 5'-AGACTGCGTACCAATTC-3' 5'-GATGAGTCCTGAGTAAC-3' Primer designation Selective bases Primer designation Selective bases

El' AAC" Mr CTA E2 ACC M2 CTG E3 ACT M3 CAC E4 AGC M4 CAG E5 ACA M5 CAT E6 ACG M6 CAA E7 AGG M7 CTT E8 AAG M8 CTC E9 AGT M9 CCA ElO AAA MIO CCC Ell AAT Mil CCG E12 AGA M12 CGT E13 ATA M13 CGA E14 ATC M14 CGC E15 ATG M15 CGG E16 ATT M16 CGT

'EcoRl primer. •"The three-letter sequence designates the selective bases that were added to the 3' end of the core sequences. The first nucleotide A in the three-letter sequence of the £coRI primer and the first nucleotide C in the three-letter sequence of Msel primer were used as selective base in the pre-selective amplification. ° Msel primer.

AFLP protocol

The procedure was performed according to the protocol provided by GIBCO/BRL and described previously (Yu and Wise 1999). AFLP primers and their sequences are listed in

Table 1. The initial primers used were derived from the large-genome kit supplied by

GIBCO/BRL Life Technologies, Inc. (Gaithersburg, MD) (designated El to E8 and Ml to

MS). Supplementary primers (E9 to E16 and M9 to M16) were designed by Fusheng Wei of 87 the Wise lab and were synthesized at the ISU DNA Sequencing and Synthesis Facility

(Ames, lA).

Bare-l-like retrotransposon, sequence-specific amplification polymorphism (S-SAP)

Procedures to analyze retrotransposon sequence-specific-amplification polymorphism were as described in the AFLP protocol with minor modification. A Bare-l-like oligonucleotide (Bare-l-like-LTR: 5'-CTAGGGCATAATTCCAACA-3') was end-labeled by combining 10 |il [^^pj-y-ATP (3000 Ci/mmol), 10 |J.l 5x T4-kinase buffer (0.25 M Tris-

HCl PH 7.5, 0.1 M MgC12, 50mM DTT, 5mM spermidine) and 18 ^il Bare-l-like-LTR (5 liMol), 2 |il kinase (25 units) and 10 ^il sterile distilled H20 (Waugh et al. 1997). The end- labeled Bare-l-like LTR was used in the selective amplification in combination with either

A/^el-AFLP primers or f'coRI-AFLP primers.

Data analysis

Polymorphic amplified DNA fragments detected by AFLP primer pairs were scored separately from the top of sequence gels to the bottom. The loci detected by these DNA fragments were designated according to the names of the primer pairs, the position of the fragments on the sequence gel and their parental specificity (fragments specific to C.I. 3815 were labeled as "a"; fragments specific to C.L 1994 were labeled as "b"). For example, if a fragment was the top polymorphic band and amplified by a primer combination of E-AAC

(number 1 EcoKl primer) and M-CGT (number 16 Msel primer) in C.I. 3815, the locus detected by the fragment was called "ElM16-la". The same method was used for designating the Bare-1 retrotransposon-based S-SAP markers.

Mapping data were entered into Map Manager V2.6.6 (Manly 1993; http;//mcbio.med.buffalo.edu/mapmgr.html). To build genetic linkage groups, command

"rearrange" in Map Manager was used to sort the AFLP markers. Then additional markers were inserted into the linkage groups by command "link", combined with double-crossover 88

analysis. Loci were placed in locations that generated the lowest reconnibination frequency

and the least double crossovers. The data were further prepared for MapMaker by using the

Dupont de Nemours and Co.; Lander et al. 1987). All map distances were calculated in

MapMaker using the Kosambi correction. Markers were grouped into subsets at a LOD of

6.0, and distantly linked markers were placed with a LOD of 3.0. The diploid oat data were

coded for Rl-self analysis, and grouped into subsets with a LOD cutoff of 6.0.

Results

A high frequency of amplified fragment length polymorphism (AFLP) is detected in diploid Avena

AFLP marker technology facilitates efficient DNA fingerprinting and analysis of a large number of polymorphisms via polyacrylamide gel electrophoresis. Two hundred and fifty- six pairs of AFLP primers were tested on the diploid kwna parental accessions, A. strigosa

(C.I. 3815) and A. wiestii (C.I. 1994). Out of 16,140 amplified DNA fragments that were identified via this initial survey, 1475 were polymorphic. The total number of amplified fragments as well as the number of polymorphisms varied among the AFLP primer pairs.

Table 2 lists forty-one AFLP primer pairs that revealed at least 10 polymorphisms between the two diploid lines. The combination of the EcoRI primer E-ACT and the two Msel primers M-CAT and M-CTA detected as many as 18 polymorphic amplified fragments. In contrast, the primer pairs E-AGT and M-CGG, E-AGT and M-CCA, or E-AGG and M-AGT detected both very few amplified fragments or polymorphism between the two diploid Avena accessions (data not shown). 89

Table 2. Selected AFLP primer pairs and the polymorphism between the two diploid Avena parents, A. strigosa and A. wiestii. Number of Nmnber of polymorpliic polymorphic Mse\ amplifled Mse\ amplified £coRI primer primer fragments £coRI primer primer fragments E3-ACT M2-CTG 18 E15-AAT M4-CAG 12 E3-ACT M5-CAT 18 E5-ACA M3-CAC 11

E5-ACA Mll-CCG 16 E6-ACG M6-CAA 11 ElO-AAA Mll-CCG 16 ElO-AAA Ml-CTA 11 El-AAC M6-CAA 15 ElO-AAA M9-CCA 11 E13-ATA Mll-CCG 15 E3-ACT M7-CTT 11 E13-ATA M3-CAC 14 ElO-AAA M3-CAC 11 E2-ACC M9-CCA 13 Ell-AAT M9-CCA 11 E16-ATT M9-CCA 13 E16-ATT M16-CGT 11 ElO-AAA M2-CTG 13 E5-ACA M8-CTC 10 E13-ATA M15-CGG 13 E6-ACG M5-CAT 10 El-AAC M4-CAG 12 E3-ACT M8-CTC 10 El-AAC M4-CTC 12 E2-ACC M6-CAA 10 E3-ACT Ml-CTA 12 E13-ATA M2-CTG 10 E3-ACT M12-CCT 12 E13-ATA M4-CAG 10 E4-AGC Mll-CCG 12 E3-ACT M13-CGA 10 E5-ACA M6-CAA 12 E2-ACC M13-CGA 10 E9-AGT Mll-CCG 12 E15-ATG Mll-CCG 10 Ell-AAT M5-CAT 12 E14-ATC Mll-CCG 10 E13-ATA MI3-CGA 12 E16-ATr M12-CCT 10 E14-ATC M2-CTA 12

Mendelian inheritance is observed among a majority of AFLP markers in the A. strigosa x A. wiestii RI population

Forty primer pairs were used to map amplified fragments in the diploid Avena RI population. Three hundred and seventy-two AFLP markers were generated. Subsequent to data entry into Map Manager, the inheritance and genetic linkage of AFLP markers were 90

analyzed. Eighty-four percent of the AFLP markers displayed inheritance not significantiy

different (P=0.05) from a 1:1 ratio. Fig. 1 illustrates the tj^ical inheritance pattern of single

alleles for dominant and co-dominant AFLP markers.

Bare-l-based, S-SAP analysis reveals widespread distribution and heterogeneity of

retrotransposon-like sequences in the diploid Avena genome

To survey the distribution of retrotransposons and its potential usage in DNA genotyping

of diploid Avena, all thirty-two combinations of a Bare-1-like-LTR with random AFLP

primers (including 16 £coRI primers and 16 Msel primers) were surveyed (Table 1). An average of 50 DNA fragments was amplified by each primer-pair combination. A polymorphism rate of twelve-percent was detected as compared to 9.2% detected by standard

AFLP. Eleven primer pairs that exhibited the highest level of polymorphism were tested on the complete RI population. Similar to the standard AFLP markers, 83.75% of the eighty S-

SAP fragments that were mapped displayed segregation patterns among the RI population not significantly different from a 1:1 ratio (P=0.05).

Bare-l-like retrotransposon-specific amplification revealed possible transposition in the diploid Avena genome

Retrotransposons play an important role in genome evolution (Flavell et al. 1992; Voytas et al. 1992). However, it is often difficult to analyze the activities of the various retrotransposons due to a low transposition frequency. Retrotransposon-specific amplification via AFLP is an ideal method to detect these movements because genome-wide scanning can be visualized on a series of autoradiographs. Recent transposition and insertion events of retrotransposons can be postulated if novel amplified DNA fragments present in one or more individual RI lines in die population but not in the parents. To test this assumption, the autoradiographs of the eleven Bare-1 based S-SAP mapping gels were examined for these novel amplified DNA fragments. Forty-nine such fragments were 91

observed out of seven hundred Bare-l-like amplicons. As illustrated in Fig. 2, these

amplified DNA fragments occurred randomly on individual RI lines but not on the parents.

Fig. 3 shows the segregation of Bare-l-like, retrotransposon-specific fragments amplified by

the Bare-l-like-LTR and AFLP E-AAA primer pair. The loss of fragment Bare/ElO-2

appeared to be coincident to occurrence of new fragments at the positions of Bare/ElO-l

and/or Bare/ElO-3. In contrast, the amplified S-SAP markers, Bare-E10-5a and Bare-ElO-

6b, displayed a stable segregation not significantly different from a 1:1 ratio (P=0.05). For comparison, the autoradiographs of the standard AFLP mapping gels were also examined for these phenomena. No such novel fragments were found.

To determine whether these novel amplifications were the result of transposition, the coincident fragments Bare/ElO-1, Bare/ElO-2, and Bare/ElO-3 (Fig. 3), were cloned and sequenced. As illustrated in Fig. 4, Bare/ElO-1 is identical to Bare/ElO-2 except for a 19 nucleotide (nt) insertion at 3' end of Bare/ElO-2 sequence and Bare/ElO-3 is 96% identical to

Bare/ElO-2 but has a 76 nt deletion at 5' terminal of the sequence. This result suggested that those DNA fragments may be due to insertion or deletion invoked by transposition of retrotransposons. Altematively, this could also be due to retrotransposon-induced, unequal recombination. To verify the above result, PGR primers specific to the deletion or the insertion were designed. These primers were tested on the parents and RI lines with these novel fragments. Unfortunately, an identical size product was amplified in both the RI that possessed the novel fragment and those that did not. This observation is likely due to the high copy number of the Bare-1-retrotransposons (Suoniemi et al. 1996).

Anchoring the AFLP and S-SAP markers on the diploid Avena linkage groups with known grass-genome RFLP probes

To position the above described AFLP and S-SAP markers on the A. strigosa x A. wiestii linkage map, each linkage group was anchored with known grass-genome RFLP markers. RI population

Fig. 1. Autoradiogram obtained with AFLP primer pair of E4 and Mil, showing the segregation of 2 typical AFLP markers (designated by arrows) on the A. strigosa (C.L3815) x A. wiestii (C.I. 1994) RI population. E4Mll-2a is a dominant marker while E4M1 l-3b is a codominant marker. Rl population 0) x-O) 00 a> CM C0'*^-l0<0f^000>O'r-CNJC0'^l0<0r^C0a) Or- C\I COCNJCO'^lOCDr^OOOO^CNJ co^t E§- ® COT- oo OOOOOOOt-t-t-t-v-t-t-t-t-t- CN CN CN CNmiOlOLOlOLOlOLOCDCOCO CO CO 0)0'to oooooooooooooooooooooooooooooooooooo oo 00 OOOOOOOOOO oo OOOOOOOO OOOOOOOOOOOOOO 00 oo 00 OOOOOO CO OOCOOO COCOOOOOCO oo 00 •«- d-d) oo <<<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<< III(D .E c u) c -ii 2) S c CO Q_a Bare/E5-1 Bare/E5-2 Bare/E5-3 U)VO Bare/E5-4 'v; Bare/E5-5

Fig. 2. Autoradiogram obtained with Bare-1-like LTR primer and AFLP primer E-ACA demonstrating the segregation of amplified Bare-1 like fragments on the A. strigosa (C.I.3815) x A. wiestii (C.I. 1994) RI population. Bare/E5-1, Bare/E5-2, Bare/E5-3, Bare/E5-4, and Bare/E5-5 are five Bare-specific fragments present in some RI lines but not in parents. (/)

E <» RI population D)^W •t2 CLg C O T- CMCOOJOT-CMCO-^ LO CO CO Oi o CM OOOOOOO OOT-T- T-T-CNJCOCOCOCOCO CO CO CO CO CO Tt, Tt- E ooooooo o ooo ooooo o o o _ _ _ O oooo— — — a. C3DCOOO COOOOOOO 00 COOOCO COOOOOOOOOOO COCO 00 CO CO CO OO OO CO GO < OO <<<<<<< ^ - < < < < CO I CO o *o Bare E10-1 Q.(D (/} Bare/E10-2

2CO m

Bare-E10-5a Bare-E10-6b

Bare/El 0-3

Fig. 3. Autoradiogram obtained with Bare-1-like primer and AFLP primer E-AAA and showing novel DNA fragment segregation pattern revealed by the AFLP technique (pointed by arrow) on the A. strigosa (C.I.3815) x A. wiestii (C.I. 1994) RI population. Both DNA Fragment A and Fragment C were amplified on the DNA of neither parents but on some individual lines of the RI populations. Fragment B occurred in both parents but disappeared on some individual lines. Bare-1 primer 19 nt insertion I I iiiiiiiiiiiiiiiiiiiiiHiniiiiiiiiiiiiiimiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiifimimiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiHiiiiiiiiiiiiiiiiiiiiiiiimiiiHiiiiiiiiiiiiiiiiiii Bare/E10-1

liiiiiiiiiiiiiiiiiiiHiiiiiiiiiiiiiiiiiiitiiiimiiiiitiiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiiiimiiiiHiiiiiiiiiniiiimuiiiiiiiiiiiiiiiiiiHiiimiiiiiiiiiiiiiiiH | Bare/E10-2

iiimiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiimiiiiiiiiitiiiitiitiiiiiiiiimiiiiiiiiiiiiiiiiiiiiiiiiHiiiiiiiiiiH | Bare/E10-3 t t 76 nt deletion AFLP primer

Fig. 4. Sequence comparison of three coincident Bare-l-like fragments amplified with Bare-l-likeprimer and AFLP primer E-AAA. Bare/ElO-2 is the fragment that was amplified in both parents and most of the 100 RI lines. Compared to Bare/ElO-2, Bare/ElO-1 has an identical sequence except for a 19 nt insertion at the 3' end of Bare/ElO-2 sequence and Bare/ElO-3 is 96% identical but has a 76 nt deletion at the 5' terminal of the sequence. 96

Table 3. Linkage group, polymorphism, and restriction enzyme survey of anchor probes.

Linkage Enzyme

Probe* Group'' EcdS. Kpnl £coRI Hindu Oral Reference V I

isul357 F + + + + + Rayapati et al. 1994a isul776 B n n n n + Rayapati et al. 1994a

isul6S6 D - + + + + Rayapati et al. 1994a

isul686 D - + - + + Rayapati et al. 1994a

isul553 F + n + + + Rayapati et al. 1994a isu2203 E + + n + + Rayapati et al. 1994a isul 946 G + + + + + Rayapati et al. 1994a

isul839 C + + + + + Rayapati et al. 1994a

isu2263 D - + - + + Rayapati et al. 1994a isul876 B + + + + + Rayapati et al. 1994a

isul 651 D + + - + - Rayapati et al. 1994a

cdo36 C + - + - - Van Deynze et al. 1995b

cdo38 G - - + - - Van Deynze et al. 1995b cdo520 AE + + + + + Van Deynze et al. 1995b

cdo400 E + - - + + Van Deynze et al. 1995b

bcd450 EFG - + + - - Van Deynze et al. 1995b

cdo395 AB + + + - - Van Deynze et al. 1995b

cdol380 G - - - - + Van Deynze et al. 1995b cdol502 C n n n + n Van Deynze et al. 1995b

cdo545 D - - - - + Van Deynze et al. 1995b 97

Table 3. (continued)

isul719 A + + + + + Yuetal. 1996 isu2192 A + + + + + Yuetal. 1996 isul410 A + + + + + Yuetal. 1996

isull46 A + + - - + Yuetal. 1996 isul774 A + + + + + Yuetal. 1996 isu2191 A + + + + + Yuetal. 1996

isul785 A + - - + - Yuetal. 1996

isu2117 A - - + - - Yuetal. 1996 isu2232 A + + + + + Yuetal. 1996 isu916 A + + + + + Yuetal. 1996

bcd98 A - + - - - Yuetal. 1996 cdo580 A + + + + + Yuetal. 1996 cdol473 A + + + + + Yuetal. 1996

cdo708 A - + + - - Yuetal. 1996 mwg060 A + + + + + Yuetal. 1996 mwg068 A + + + + + Yuetal. 1996

mwg075 Unknown - + + + + Yuetal. 1996

* Polymorphism were not detected by the following anchor probes: cdo017(D), cdo078(D), cdol05(G), cdo244(B), cdo328(C), cdo460(C), rz244(AC), cdo89(D), cdo516(DG), cdo962(D), cdo941(AC), cdol387(F), cdo470(F), cdol328(B), cdo692 (D), isul313(G), and isull88(B). The uppercase letters in parenthesis following the probe designation are their respective linkage groups, n indicates that the data is not available. '' The terminology of linkage groups was specific to their source maps indicated in the table. These linkage groups may not be identical. For instance, linkage group B in the ISU map (Rayapati et al. 1994a) is not the same linkage group B in the Cornell map (Van Deynze et al. 1995b).

Thirty-seven anchor probes were selected and tested as shown in Table 3. Probe selection was based upon the distribution of these RFLP markers on seven linkage groups of diploid

Avena. Twenty-four probes were selected from the anchor probe kit for comparative mapping of grass species, kindly provided by Dr. Susan McCouch (Cornell University) (Van

Deynze et al. 1998). Another 13 were selected based on their positions on the original Fj map of A. strigosa \A. wiestii (Rayapati et al. 1994a). Nine out of 24 anchor probes revealed RFLPs between A. strigosa and A. wiestii, whereas 11 of the 13 ISU probes detected 98 polymorphism with the enzems tested (Table 3). Also integrated into Table 3 are RFLP markers from a previous study targeted to Group 1 homoeologous regions in Avena,

Hordeum, and Zea mays (Yu et al. 1996). Linkage groups were established by the approach described in the Materials and Methods and are shown in Fig. 5. Five hundred and nine makers were mapped on seven linkage groups. The designations given to seven linkage groups correspond to the seven linkage groups in the A. atlantica xA. hirtula map (O'Donoughue et al. 1992; Van Deynze et al. 1995b).

Clustering of markers leads to the formation of highly saturated linkage blocks

The genetic markers were not uniformly distributed among the seven linkage groups (Fig.

5). Clustering of markers creates regions of highly saturated linkage blocks. Table 4 lists these linkage blocks, their distribution on the diploid Avena map, marker densities, and representative DNA markers. These linkage blocks include almost three-fifths (282/514) of the markers, but less than one-third of (1054 cM/3480 cM) coverage of the map. The average marker density in these blocks is 3.74 markers per cM compared to an average of 6.7 cM on the entire map. One particular interesting linkage block, B2, consists of five distinct crown-mst resistance genes and the tightly linked AFLP derived STS (sequence tagged site) markers, Agx4,Agx7, and Agx9 (Wise et al. 1996; Yu and Wise 1999). Although S-SAP markers are generally uniformly distributed in the map, eight of these markers mapped in a cluster on linkage group E (block El). A cluster of resistance gene analogs was also positioned in the linkage group but in block E2. Compared to the distribution of other genetic markers on diploid Avena, the distribution of the S-SAP markers is relatively uniform. The S-SAP markers can be found evenly within the saturated linkage blocks and within other sparsely populated map regions as well. Fig. 5 Unk^ group A (94 DNA maifceis and 575.8 cM): Xcdo580, Xmwg068, Xcdo395A, and

Xcdo520A conespond to maricers previously mapped to group A on the ISU A. strigosa x A. wiesdi and the

Cciri^A.atlanticaxA.hirtida 100

Bare-E12-8a 5.7 xisunee xmwgoes

15.8 XcdoU73b 11.1 E5M6-12a 10.2 E13M11-15b 8.9 Bare-M15-2a 11.1 E13M15-8a E5M11-2a 9.1 E11M14-2a E1M6-7a E5M6-2a E16M9-1a E12M12-10b 12.7 Bare-E12-1b E16M11-4a 10.7 E16M9-6a Bare-El 0-2a E3M2-9bE11U10-10b E5M11-5a 13.6 E13M13-2a

17.1 E15M11-2b Ei3M3^b Bare-E2-4b E4M4-1b Bare-E14-7a Bare-E14-6a 18.4

E5M6-8b E3M5-6b E13M3-2b xdo39Sb 13.4 E10M11-13a E11M5-9a E1M3-9b 4.9 E13M15-1b E10M2-1b E5M11-4b 14.3 E13M15-9b E16M9-5b XcdoTOS 4.2 E14M11-9b E3M5-4b 3.7 xcdosao 4.7 Bare-M13-6a 7.A E9M11-10b E3M5-14b 7.9 E4M11-11b

E6M14-1 ESM11-8a E3M5-3b 9.7 E11M10-7a ESM15-1b£.|3f^.,3.7a E15M11-8b 2.6 4.4 E13M15-10b 6.9 E3M12-6b E1M3-1a 3.9 XISU2232 E3M1-8b 7.0 Bare-El 0-3b E12M12-2b 8.3 E13M11-12a xisuUlOe E11M15-6a a E1M4-8b 5.1 Bare-Mi 5-5a E7M4-1a Ei6M11.11a xisuUlOb suUlOb 13.8 E16M9-8b xtsum* 5.9 E10M11-6a Xmwgoeo Xtsu916 E2M4-3b Bare-E5-4a Bare-E12-9a 1-8 3:9 Xlsu1719 5.1 Bare-E14-1a E2M9-4a 9.6 Xcdo1473a 5.4 E3M12-1b E2M4-5b 5.0 1.9 E16M11-1b E13M13-3bE5viii^b E11U9-4a 2.8 E7M4-Sa Xcdo1473 E15M11-5b 10.7 E1M6-2a Bare-E12-8a

Linkage group A Fig. 5 (continued), linkage group B (82 marioos and 461.9 cM):Xcd(9595fl andX&ccH^conespondto niaikmpie\douslym£5)pedtog^)iq)BonflieCcjmeUA.flrfla«ribflxA./a7Mzm^. The Pea resistance- gene cluster is linked to RFLP marker XwM2i92 on this linkage group. 102

XISU2192

20.4

Agx7 4.3 0.6 i l Pc81 PC82-290 Pc82-2T6 Pe83 "•5 li Pc84 pcBSAgx4 Agx9

11.3

E12M12-3a Operon C18

E1SM11-6b 25.0

E3M5-Sa E3MS-7a E3M5-1a 9.4

E10M1S-1a Bare-E14-2b

E10M16-10a 23.5

E15M11-7a E3M12-5b E3M12-2b E14M11I-fib E10M16-12a 2.5 E13M12-5b ^•'•'Mis-la E10M16-13b E2M9-7a 8.7 Bare-E3-1b Xcdo395a E9M11-2a E10M16-1b*6crf98*;si/2ff7l BaFe-E14-4b 9.8 E3M2.14bE3M2-15a^^3^^^^^ E13M12-7a 6.0 E10M16-10a E1M3-2b E16M11-2a 8.5 E13M11-13a E13M11-14b E10M16-2b ^^'°^-'^^Xcdo1T8S 9.5 Bare-E16-5a Bare-E1-2a Bare-E1-1b E12M12-6a Bare-M13-8b M E16M9-1^.,5j^g.2a

E10M1&-Sa 18.7 E5M16-1a E15M4-7b E13'*^11-®®*/su2f9fa E5M15-3a E5M6-9b E4M4-4b Xlsu2191c Xlsu2191d cinMii

E1SM4-8b E3M5-9a E14M11-12a E14M11-7aBare^15-3a Bare-E15-6a E3M2-8a 25.7 iJjjiJ^aa E13M11-3a

XISU2192 E3M5-2a

Linkage group B Fig. 5 (continued). Tinifagp group C (49 markers and 383.8 cM):Xisul839 andXcdo36 conespond to

markers previously mqjped to group C on the BUA. strigosaxA. vweaf/and tiie Cornell A atlanticaxA.

hirtidavnaps. 104

E1M6-1a

E1M3^ E4M11-8a Biiui-Lih.r-«.o t.. E16M11.6a E1M4-1bE16M9-7bE3M5-8a E1M3-7b E1M4-3a „ E1M3-8a E11M10-1a E1M3-5b E1M3-3a

E12M12-9b E2M9-6a

E4M4-9b Bare-E2-2a EgM4-4a

E13M15-4b E14M11-3b E2M4-9b

Bare-M8-9a

Bare-E12-4a

E3M2-1b E10M16-9b

E14M11-10b

E13M12-2b

E9M4-1a E2M9-1a

E13M11-1b E5M11-7b xisuim

Xcdo1S02

Bare-M8-1a E3M5-12a E3M2-11a

Bare-M13-1a

Bare-M13-2a

E2M9-5b E13M11-4a E10M11-5b

Bare-E2-3b Bare-El 4-5b E3M5-11bE12M12-5b E15M4-12b

E10M15-2b

Linkage group C Fig. 5 (continued). Tinkagp group D (41 markeis and 332.9 cM): Xisull46, Xisul651, and Xcch545 cxjn^e^nd to maifceis previously mqqsed to group D on the ISU A. strigosa x A. wiestii and the Cornell A. atlanticaxA.hirtida s CM LU nj^ SI" si 5 UJ6 uiiu C a r4 to ^ A (0 (O (0 jn •JIX? Ill I CM '»• d> s5 3 si S a S i iiiiii i 1 1 n i i CM Si 1 i .e CD liJ III liJU PQ lUQQ lij UJ ijjuj lu Ui UJ Ui CD UJ UJ UJ uiui UJ UJ IJJ 2 X X O)

^inqqr)

i. strigosa x A. wiestii F2 m^. One cluster of RGAs and one cluster of Bare-1 specific markers are positioned on this linkage group. RGA-LM-850 was also mapped in this linkage group and is closely linked to a crown-rust resistance locus, R264B-2. 108

E1M4-5a

20.3

E14M2-3b 12.9 E13M12-8b 1.2 E10M11-1a E1M4-6b E13M15-7bE10M16-3b E4M11-9E9M11-8b E4M11-€aE4M11-7bE4M11-6b 16.4 RGA-LM750 RGA-IM950 RGA-LMmORGA-C3 BaFe-M15-4b ii ^Af9RGA^ RGA-H3B^1SM4.1U E16M9-9bg,4j^2-2a 3.9 E2M4^b 7.3 E12M12-1b E1M3^b 6.9 E13M15-3b

S^^FLI-RES-2a 19.4 E2M9^b E16M11-9a E3M2-4b HSA-LM8S0 14.3 E9M4-9a 5.0 Bare^8-4a R246B-2 9.6 Bare-M15-12a 6.1 E10M11-7a ei1«5^a 15.6 E1M6^ E11M15-4b Bare-E5-3b 10.7 Bare-E12-3b Bare-E12-7b E3M2-6b E9M11-4b 3.2 E9M11-5b 7.0 XcdoSS EIIMIO-Sb 8.5 E2M4-4a Bare-E10-5a 3.9 E15M4-5b 6.1 Bare-E3-2a E15M4-€a Bare-E12-6b 6.0 Bare-M8-Sb 1.9 E5M6-Sb E16M11-5a1 Bare-M1S-6b 6.9 Bare-E14-3a E4M11-1a 12.8 E3M1-6b 4.9 E2M4-1a Bare-E1&-4b 6.1 Xcdo400 Bare-E10-7a E3M12-7b 14.3 ESM6-7a ESM1S-4b 12.1 XISU2203 7.1 =1= E9M4-8b E3Mi.3b 1.9 E13M12-4b E15M4-7a 11.4 E10M2-2a E12M12-7a E16M9-4a 4.0 E3M2-2b 0.9 E2M9-9b E3M1-8b 7.1 E13M12-1b Bare-EIO^b 19.6 Bare-M8-6b E13M3-1b E11M10-6b 11.7 E9M11-3a 5.7 E1M6-3b 7.9 E5M1S-7b E10M16-14a XmwgOTS 10.5 E1SM4-4b E11MS-8b 9.2 E9M11-9b 3.1 Bar&M15-7a E13M12-7b 5.4 E1M4-4b Bare-M15-9a 8.1 E1M4-5a E15M4-1b

Linkage group E Fig. 5 (continued). link^ group F (60 mariceis and 434.7 cM): Xisul553, Xisul357 and Xcdo38 correspond to markers previously mapped to group F on tiie ISU A. strigosa x A. wiestii m^. Xisu2263 and the closely linked marifier, Xcdol250, cone^nd to madceis previously m^jped to group D on the A. strigosa

X A. wiestii but weie located on group F of the Ctomell map. 110

Bare-M13-7a

E9M11-7a E11M14-3a E11M5-7b

E15M11-3a E5M16-3b E16M11-Sa2 E3M1-10a E16M9-11b E16M9-13b XISU1357 E15M4-9b

E7M4-8a E12M12-8b Bare-E5-1b

E11M5-7b

E3M2-7a E11M5-4b Bare^15-1Sb

E11MS-Sa E11M15-7b E11M10-9b E3M2-12b E13M15-5a

E16M11-3a E3M1-7a isu2191b

E11M5-1a XISU1S53 E10M11-4a

E9M4-6a

E7M4-6a E2M4-10a

E12M12-4a

E2M9-2b2

ESMie^b E13M15^b

Bare-A415-14a ESM11-9b E15M11.1a^2M9-2b XISU2263 Ei5Mii^b E3M5-10b E14M11-4a E13M13^b Xcdo38 E13M11-8a E13M11-8b E3M1-5a E2M4-8a Bare^16-3b Bare^8.2b E7M4-3b El5M11-9a Bare-E1-4a

Bar&M8-7b

Linkage Group F Fig. 5 (continued). Link^ group G (81 maricers and 592.1 c^O: Xbcd540, andXcdol380 cone^nd to madsers previously m^jped to group G on flie Cornell A. atlantica xA. hirtula map. oob>b)

-» 0> WW -A -i uic*>ro -k tn cn-* -i hj cnw-^ Co 09N o or' •-» b ^ f*® b)OJ ^(0(0 Nfajo r^^bo N (DOIOaXD ui I^iofo b

mm m m rom m m rom 00u 6>m a>CO -Am « oo m m m m m>< com S 2." i §i g ^ u i Scn I 1 i§ IsJ & ^ ^ S51 ri lb rb So* ill ^ cr^ ItI i» 1*-* o- ^ a> yjk. O" o* V " I ^ mf V* & S lo »& is O" m ni_ m i 11 t i 70) S 3 u

•A i>tt (Q o* S§ 8 C ^1 "O to O ro roisj -»• o> -k M o» o» cu (O o ^ k> b» bo ^ LacoIOO^W5'^r^P

m m H m mm 00 m m m m S S ii 9 9 ii I ii |is 99 -k jo Nl rj •k -A I, O) 0} o* 0*0) S t ro U (Jl -k 0) m m SQ> fi)ai ro o 11 Q. tto S'S' m & 113

Table 4. Representative markers and map locations of the linkage blocks on thei4. strigosa x A. wiestii RI map.

Number of Avena linkage Density of markers Linkage block markers group (Marker number/cM) Representative markers Al 46 A 4.7 Xcdo395, XisuI766, Xcdol473C, zniXmwgOdS

A2 33 A 1.98 XcdoSSO, Xcdo708, XmwgOdO

B1 49 B 2.37 Xbcd98,Xcdol785, md Xisu2192

B2 8 B 1.46 The Pea resistance gene cluster

CI 15 C 3.45 A cluster of AFLP markers El 36 E 5.13 Xcdo36-E, Xcdo520-E and Xisul786 and a cluster of Bare- 1-like, S-SAP markers

E2 11 E 1.61 A cluster of resitance-gene analogs

F1 23 F 3.9 Xcdo38-¥ and Xisu2236-F

G1 34 G 4.53 Leaf sheath pubescence locus

G2 27 G 5.4 Xbcd450, Xcdol380-G

Average 28 3.74

Increased saturation clarifies previously known gaps in the A. strigosa x A. wiestii Fj

RFLP map

The classification of linkage groups on the diploid Avena RI map is generally consistent with the F2 RFLP map of A. strigosa x A. wiestii (Rayapati et al. 1994a). However, due to the 114

increased density of markers, several linkage groups were rearranged. Two loosely linked

linkage groups on the Fj group A were grouped into the current RI groups A and B

respectively (Fig. 5, Linkage group A and Fig. 5, Linkage group B). The top half of the

original Fj group A is part of the newly established RI group A and the bottom half is now

part of current group B. This is because the previously classified group-A anchor markers

Xmwg060, Xmwg068,Xisu916, Xcdo580, and Xcdo395b were positioned on the newly

established group A. Xisul766 and Xisul876, two RFLP markers on the top of the original Fj

group B, were integrated into the newly established groups A and E, respectively. Xisul766 co-segregates with Xmwg068 on group A and Xisul876 is tightly linked to Xcdo520b and

Xcdo36 on group E. This result is consistent with the fact that Xbcdl549 which was tightly linked to Xisul876 on the original Fj group B is closely linked to Xcdo520b, an RFLP marker on group E on the A. atlantica x A. hirtula map (Van Deynze et al. 1995b). The other rearrangements can be evidenced on linkage group D and F. The top linkage block of original

F2 group D was classified as a part of the newly established group F. This is because

Xisu2263 on the original F2 map was positioned on group F, closely linked to Xcdo038 (Fig.

5, Linkage group F). Furthermore, on the original F2 group D, Xisu2263 was closely linked to

Xbcdl250 which is closely linked to Xcdo38 on group F on the A. atlantica x A. hirtula map

(Van Deynze et al. 1995b).

The anchored AFLP map from the A. strigosa x A. wiestii RI population is consistent with diploid Avena map based on A. atlantica and A. hirtula

The anchored AFLP map based on the RI population from A. strigosa and A. wiestii shows consensus with another diploid Avena map based on a cross between A. atlantica and A. hirtula (Van Deynze et al. 1995b). If more than two anchor markers were mapped on the same linkage groups in the two populations, the alignment and order were identical. For example, Xmwg068-Xcdol743-Xcdo580 on linkage group A, and Xcdo400-Xcdo36- 115

Xcdo520b-Xisul876 (Xbcdl549) on linkage group E are identical between the two Avena

maps. If only two anchor markers were mapped on one linkage group, the same linkage relationships were detected between the two maps. Xcdo545 andXisul 146 (Xcdol467) were

mapped on linkage group D. Xcdo38, Xisu2263 and its cosegregating marker Xbcdl250 were

mapped together on the bottom of group F. Xcdol380 and Xbcd450 were mapped together on group G.

A cluster of plant-disease resistance-gene analogs are positioned on the saturated map

Several plant disease resistance genes have been cloned and molecularly characterized

(Michelmore 1995; Lawton 1997). These genes, isolated from a variety of plant species, condition interactions with a broad range of pathogens, including bacteria, fungi, viruses, and nematodes. Interestingly, most of these genes encode proteins that share conserved amino acid motifs. PGR primers designed from these conserved motifs have been demonstrated to be an effective way to amplify resistance-gene analogs (RGAs) (Kanazin et al. 1996; Leister et al. 1996; Yu et al. 1996; Meyers et al. 1998). The primer pair, LM637 and LM638, developed according to the conserved sequences of three cloned plant resistance genes,

RPS2, N and L(5, (kindly provided by Dr. R. Shoemaker, Department of Agronomy, Iowa

State University), was used to survey the G.I 3815 and G.I. 1994 parents for genetic mapping.

Five polymorphic fragments were detected by PGR amplification. Among these, RGA-

LM850, a fragment of 850 nt, mapped on the top of linkage group E. The next three fragments, RGA-LM750, RGA-LM950, RGA-LM1200, were positioned at another locus on linkage group E. RGA-LM2000 mapped on linkage group G, linked to AFLP marker

E4M1 l-2a and RFLP marker XisuMlOa.

The LM637 and LM638 primers were also used to amplify resistance-gene analogs from

D526, a crown-rust resistant line of hexaploid oat. These amplified fragments were ligated into the pGEM-T vector (Promega, Madison, WI) and a mini-library consisting of 192 116 colonies was established in two microtitre plates. Four distinct polymorphic clones, RGA-

C3, RGA-G4b, RGA-H3b and RGA-F9, were mapped via Southern hybridization of the

//mdin digested DNAs of the RI population. RGA-C3 cosegregated with a locus previously detected by RGA-LM750, RGA-LM950, RGA-LM1200. The other three polymorphic clones, RGA-G4b, RGA-H3b and RGA-F9, were positioned 2.4 cM from the locus detected by RGA-C3, RGA-LM750, RGA-LM950, and RGA-LM1200.

To determine whether these cosegregated fragments were related to each other, their nucleotide sequences were analyzed via pairwise BLAST searches. Partial similarity was detected among some of the sequences as shown in Table 5. RGA-LM850 shared a DNA fragment of about 300nt with RGA-F9 and RGA-LM1200 that was 80% similar. RGA-F9 shared two small fragments (130 nt and 57 nt long) with RGA-H3b that was over 81% similar. Results from these analyses indicated that all eight fragments were unique.

Table 5. Significant BLASTN similarity among resistance gene analogs (RGA) in diploid Avena Sequences of a high similarity' RGAs compared Length (nt) Position (nt) Percent similarity RGA-LM850 RGA-F9 281 3-283 76

RGA-LM1200 301 1-301 78

RGA-LM1200 RGA-F9 119 165-283 80 66 3-68 87

RGA-C3 RGA-H3b 130 8-137 81

57 173-229 85

' The resistance gene analogs, RGA-LM750, RGA-LM850, RGA-LM950, RGA-LM1200, RGA-G4b, RGA- H3b, RGA-C3, and RGA-F9, were subjected to pairwise BLASTN comparison (BLASTN, 2.0.8). 117

To determine if the amplified fragments were similar to other cloned-plant-resistance genes, the sequences were analyzed by BLASTN and BLASTX-PIR database searches. The results of these analyses are shown in Table 6. Of the eight fragments, RGA-LM750, RGA-

LM850, RGA-LM1200, RGA-C3, RGA-G4b, RGA-H3b and RGA-F9 displayed a high level of similarity to Hordeum vulgare NBS-LRR type resistance protein (b7 and b8) genes.

These two resistance gene analogs were previously amplified by NBS-LRR-type-resistance- gene-specific primers via rtPCR using barley RNA as a template (Leister et al. 1998). RGA-

LM950 showed similarity to the A1 and CXa27 resistance-gene-family members (Song et al.

1995). Xa21 confers resistance specificity to multiple isolates of rice bacterial leaf blight pathogen, Xanthomonas oryzae pv. Oryzae (Mew, 1987; Nelson et al. 1994). RGA-LM1200 and RGA-G4b displayed a high level of similarity to RPMl, an Arabidopsis bacterial resistance gene (Grant et al. 1995). However, BLASTX-PIR search revealed a high level of similarity of RGA-LM950 to TgmS, a soybean transposable element (Rhodes et al. 1988).

Taken together, these results indicate that these amplified DNA fragments from Arena were indeed resistance gene analogs.

A second locus that determines resistance to crown rust isolate 264B is integrated into the newly established AFLP map

Crown rust, caused by Puccinia coronata f. sp. avenae, is an important fungal disease of cultivated oat. Infection types (IT) of resistant oat plants are often characterized by a hypersensitive response and are inherited as single Mendelian genes. If resistance were conferred by a single gene, 96.75% of the lines should be homozygous for either allele by the sixth generation of selfing (at a 1:1 ratio). If, however, resistance were conferred by two

(independent) dominant genes, say Ri and R2, the ratio would be 3:1 (resistant: susceptible).

Individuals with genotypes Ri/Ri R2/R2, Ri/Ri r2/r2, and R2/R2 would all confer resistance, 118

Table 6. Comparison of RGA in diploid Avena to other resistance gene sequences Size Database Resistant gene analog (bp) Most significant similarity type Reference

Mapped by PCR RGA-LM750 750 HordeumvulgareNBS-L^ type 1' Leister etaJ. 1998 resistance protein b? gene, 2.0e-56

RPMl, &.9e-09 2^ Grant et al. 1995

RGA-LM850 850 Hordeum vulgare NBS-LRRlype 1 Leister et al. 1998 resistance protein b7 gene, 5.5e-57

//orrfewn VM/^are NBS-LRR type 1 Leister etal. 1998 resistance protein bS gene, 9.2e-17

l.le-08 2 Grant etal. 1995

RGA-LM950 950 Xfl27 member Al, (rice, 4.0e-54 1 Song et al. 1995 Xa21 member C pseudogene, 5.6e-53

soybean transposable element TgmJ, 2 Rhodes etal.1988 1.6e-43

RGA-LM1200 1200 //or£?eMffi vw/gare NBS-LRR type 1 Leister et al. 1998 resistance protein b7 gene, 9.0e-67

RPMl, 4.7e-09 2 Grant et al. 1995 119

Table 6 (continued)

Mapped by RFLP

RGA-C3 550 Hordewn vulgare NBS-LRR type Leister et al. 1998 resistance protein b7 gene, 3.5e-27

Hordewn vulgare NBS-LRR type Leister et al. 1998 resistance protein b8 gene, 8.3e-15

RGA-G4b 535 Hordewn vulgare NBS-LRR type Leister et al. 1998 resistance protein hi gene, 3.3e-65

RPMl, 1.5e-05 2 Grant et al. 1995

RGA-H3b 600 Hordewn vulgare NBS-LRR type 1 Leister et al. 1998 resistance protein b7 gene, 1.7e-29

Hordewn vulgare NBS-LRR type Leister et al. 1998 resistance protein b8 gene, 8.8e-10

RGA-F9 611 Hordewn vulgare NBS-LRR type Leister et al. 1998 resistance protein b7 gene, 2.4e-52

• A "1" represents BLASTN-GENBANK-NONST

"A "2" represents BLASTX-PIR

while an individual with a genotype of tJti i-Jvi would be susceptible. In our earlier studies,

67 resistant- and 30 susceptible- lines were detected in the Fg g A. strigosa x A. wiestii RI population in response to isolate 264B (Wise et al. 1996). This observation was in good fit to a ratio of 3:1 (resistant: susceptible), suggesting tliere were two independent genes involved.

One locus detected by isolate 264B co-segregated with the locus detected by PC54. The

RPc54 locus is positioned within the crown rust resistance Pea cluster at a LOD value greater than 5 (Wise et al. 1996). If the second locus detected by 264B were independent of Pea, IT reaction data specific to the second locus would generate false double crossovers if positioned 120 at Pea. Among 67 resistant RI lines, 13 displayed double crossovers. The occurrence of excess double crossovers in response to isolate 264B, in fact, did suggest that a second independent gene influenced the infection type of the plants. The second locus would confer resistance in absence of the dominant allele at the first locus within the Pea cluster. This would account for the 13 apparent double crossovers at Pea. Therefore, the locus within Pea was designated R264B-1 and the second independent locus was designated R264B-2. To position R264B-2, genotypes for RI lines with double crossovers (RIL 7,9,17,18, 30, 51,52,

54,65, 66,68,72, and 91) were recoded as R264B-2IR264B-2 while genotypes for susceptible

RI line would be r264B-2/r264B-2 (Wise et al. 1996). These data were integrated into our current AFLP and Bare-l-based S-SAP Map Manager file and the R264B-2 locus was positioned on linkage group E, 10 cM proximal to RGA-LM850. RGA-LM850 is a resistance gene analog similar to Arabidopsis Rpml (Grant et al. 1995) and Hordeum vulgare NBS-LRR type resistance protein gene b7 (Leister et al. 1996).

Discussion

AFLP- and S-SAP-based approaches produce an abundance of amplified polymorphisms and have been successfully used in genomic fingerprinting, gene targeting, and genome mapping (Vos et al. 1995; Becker et al. 1995; Waugh et al. 1997). Utilizing AFLP and S-SAP techniques, we established a saturated map for the A. strigosa x A. wiestii RI population.

Among 16,140 amplified DNA fragments, 9.2% were polymorphic via AFLP. Compared to standard AFLP, S-SAP greatly improved the efficiency of polymorphism discovery. A 30% increase in polymorphism was detected on the A. strigosa and A. wiestii lines with S-SAP, although a lower total number of fi-agments were amplified. This result is consistent with the report of Waugh et al. (1997), where a 25% increase in polymorphism over AFLP was detected in the barley genome. The increase in polymorphisms could be explained by the 121 effect of transposition of Bare-1-like retrotransposons such as insertions and deletions of retrotransposons, alternatively by retrotransposon-induced unequal recombination.

AFLP and S-S AP markers were stable and over 80% of them displayed normal Mendelian inheritance, illustrating that they are a rich resource of reliable DNA markers. Like RFLPs,

AFLP-detected markers are clustered on the map, leading to the formation of highly saturated linkage blocks. For example, the average genetic distance between markers on the linkage block at the top of group A is 2 cM. In contrast, there is an average distance between markers of 14 cM in a region between two blocks on linkage group A. Gap areas on linkage group B are often sparsely populated. These results are consistent with the reports from barley, tomato, and other crops (Tanksley et al. 1992; Becker et al. 1995). Unequal distribution of recombination along the chromosomes may account for such phenomena (Tanksley et al.

1992). Because recombination is more suppressed on the centromeric and telomeric regions of chromosomes, marker density appears more dense in these regions (Tanksley et al. 1992;

Van Deynze et al. 1995b). In contrast, DNA markers detected by the Bare-1 specific S-SAP technique were more evenly distributed across the Avena genome. This suggests that additional regions could be covered which, otherwise, were left out by other mapping techniques.

There is a growing body of evidence that indicates that retro-transposable elements have been involved in the evolution of plant genomes (Flavell et al. 1992; Voytas et al. 1992). The

DNA sequences of these elements, especially the 5' terminal LTR sequences, are highly conserved among several plant species such as rice, wheat, and barley (Waugh et al. 1997).

Although the Bare-l-LTR primer used in this experiment was designed based on the 5' terminal LTR sequences of barley, a large number of DNA fragments were amplified and a high degree of polymorphism was detected in diploid Avena. This result suggests that the 5' 122

terminal LTR sequence is also conserved and associated with genetic variation in the Avena

genome.

Retrotransposon based S-SAP is a potential technique to analyze the activity of retro-

transposable elements in the genome. Like AFLPs, S-SAPs can detect polymorphisms caused

by a single nucleotide mutation. However, unlike AFLP, S-SAPs utilizes the 5' terminal LTR

from the retrotransposon as a sequence-specific primer. Therefore, the possible action of

retro-transposable elements can be followed. Novel fragments were detected by the Bare-1,

S-SAP in this diploid Avena RI population. These fragments occurred randomly in some RI

lines but not in the parental lines. The ratio of the occurrence of those fragments is

approximately 7% (49/700). This result is in contrast to barley (Waugh et al. 1997) and also different from the result of our AFLP analysis in which no such fragments were amplified in

the barley genome. One of our preliminary explanations is that some of the retrotransposons

may be active in the diploid oat genome, resulting in unusual DNA fragments in the RI population. Alternatively, homologous retrotransposable elements might provide a template for unequal crossovers. To investigate these possibilities, three Bare-1 specific fragments

were cloned and the sequences were compared. These analyses indicated that the novel fragments were identical except for an insertion or a deletion. However, this could not be further verified by PGR amplification because primers specific to the insertion or deletion could not be used to distinguish the different novel fragments. One possible reason is that there may exist multiple copies of these sequences that prevented specific amplification.

Therefore, the mechanisms that caused the amplification of these novel fragments are yet to be investigated.

PGR amplification with primers designed based on conserved resistance gene sequences is an efficient approach in identifying and mapping resistance gene analogs (Kanazin et al. 1996;

Leister et al. 1996; Yu et al. 1996). Gomputer database searches indicated that all eight 123 cloned RGAs shared a high degree of similarity to either cloned resistance genes Qiall or

RPMl) (Grant et al. 1995; Song et al. 1995) or Hordewn vulgare NBS-LRR type RGAs

(Leister et al. 1998). RGA fragments were positioned at two tighdy linked map positions.

This result further supports the observation that resistance genes and resistance gene analogs tend to be clustered in plant genomes (Michelmore 1995; Kanazin et al. 1996; Leister et al.

1996; Yu et al. 1996). RGA-LM950 is unique in that it showed similarity to both the Xa21 resistance gene (nucleotide sequence) and TgmS, a soybean transposable element (amino acid sequence).

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CHAPTER 5. roENTIFICATION OF RDH AND RIH, TWO LOCI MEDIATING

HYPERSENSITIVE LESION DEVELOPMENT IN RESPONSE TO INFECTION BY

THE OAT CROWN-RUST PATHOGEN, Pl/CC/A^/A CORONATA F. SP. AVENAE

A paper to be submitted to Molecular Plant-Microbe Interactions

Gong-Xin Yu''^, Edward Braun^, and Roger P. Wise"'^'^

Abstract

Crown rust, caused by Puccinia coronata, is one of the most important fungal pathogens in cultivated oat. Avena plants with the appropriate genotype often respond to this pathogen by eliciting hypersensitive lesion development at the sites of fungal infection. Two new loci that control hypersensitive lesion development were identified in an A. strigosa (C.I. 3815) x

A. wiestii (C.I. 1994) recombinant inbred (RI) population and two backcrossed F2 (BCiFj) populations derived from RI A8059 x C.I. 3815 and A8073 x C.I. 1994. rdh, a locus controlling resistance fi[ependent ^persensitive lesion development, is recessive and mediates the response induced by crown-rust isolate 276. Rih, a locus controlling Resistance independent ^persensitive lesion development, is dominant and mediates the response induced by crown-rust isolate 290. Linkage analysis indicated that rdh is independent of resistance gene Pc82 while Rih is tightly linked with this gene. Calcofluor staining of infection sites indicated that cell death was involved in the interaction between the plant host cells and the crown-rust fungus in plants with hypersensitive lesion development, but not in

'Interdepartmental Genetics Program, ^Department of Plant Patliology, 'Com Insects and Crop Genetics Research, U.S. Department of Agriculture-Agricultural Research Service, (USDA-ARS), Iowa State University, Ames, lA 50011-1020, U.S.A. 132

plants that lack this phenotype. These results suggested that hypersensitive lesion

development and its related cell death was not essential for resistance to crown rust. Further

characterization of these genes may help clarify the relationship between plant disease

resistance and localized cell death.

Introduction

Localized cell death, the rapid collapse of pathogen-infected tissues, is a nearly

ubiquitous feature of the incompatible interaction between plants and their microbial pathogens. This reaction is termed the hypersensitive response (HR). HR has been proposed to play an important role in disease resistance in that cell death deprives the incoming pathogen of nutrients, causing pathogen containment (Heath 1980; Dangl 1995; Staskawicz et al. 1995). Localized cell death is a well-controlled genetic process. Several cell-death loci have been identified through the investigation of plant disease lesion mimics (Doke et al.

1983, 1988; Walbot et al. 1983; Wolter et al. 1993; Dietrich et al. 1994; Johal et al. 1995;

Baker and Olandi 1995; Low and Merida 1996). The Isdl (lesions simulating disease) and acd2 (accelerated cell death) in Arabidopsis are two such loci. Mutants in the Isdl locus mimic disease responses; plants develop self-propagating lesions, express PR proteins, and produce salicylic acid (SA) in the absence of pathogens (Dietrich et al. 1994). LSDl encodes a zinc finger protein that appeared to be involved in the regulation of transcription of cell death response genes (Dietrich et al. 1994). In maize, several alleles responsible for lesion mimics were mapped to the complex Rpl locus that confers resistance to the rust fungus,

Puccinia sorghi (Sudupak et al. 1993). In barley, die mlo locus is unique among resistance determinants in that it confers resistance to all known races of Erysiphe graminis f. sp. hordei, the fungus responsible for powdery mildew disease (Wolter et al. 1993). The mlo mimics disease lesions at low temperature in the absence of the pathogen. The size and 133

severity of these lesions depends on the genetic background and the mlo allele, suggesting

that an interplay between the Mlo gene product and a variety of other cellular factors

determines the extent of apposition formation (Wolter et al. 1993).

Localized cell death appears to be actively involved in plant-disease defense but these

results are not conclusive. The Arabidopsis Isdl mutants induce local and systemic

resistance to a variety of pathogens even in susceptible plant lines (Dietrich et al. 1994; Jones

1994). In contrast, Pseudomonas syringae pv tabaci Hrp" bacterial mutant Ptll528::Hrpl is

unable to elicit cell death. However, it is still competent to trigger the transcription of

defense genes in Arabidopsis (Jackobek and Lindgren 1993). The barley mlo locus mediates

resistance to E. graminis and the potato Rx gene mediates resistance to potato virus X.

However, localized cell death is not involved in either of these cases (Kohm et al. 1993;

Freialdenhoven et al. 1996). Therefore, it is necessary to further study localized cell death to

understand whether it is critical in the development of plant disease defense.

Puccinia coronata Corda f. sp. avenae Eriks., the causal agent of crown rust, is economically the most important fungal pathogen of cultivated oat. Single major genes are

usually responsible for the resistance specificity in a gene-for-gene manner (Wise et al.

1996). Two diploid oat lines, A. strigosa (C.I. 3815) and A. wiestii (C.I. 1994), have been extensively characterized for their responses to multiple crown rust isolates (Wise and

Gobelman-Werner 1993). C.I. 3815 is resistant to crown-rust infection; this resistance is often characterized by eliciting hypersensitive lesion development at the sites of fungal infection within 66 hours after inoculation whereas C.I. 1994 is susceptible. To determine the genetic basis of hypersensitive lesion development and its relation to disease resistance, a recombinant inbred (RI) population developed from a cross between A. strigosa (C.I. 3815) and A, wiestii (C.I. 1994) was investigated via infection with crown-rust isolate 276. We hypothesized that different genes control resistance and hypersensitive lesion development. 134

If so, then the hypersensitive lesion phenotype and infection type reaction would segregate

independently in the RI population. To further characterize the specific interactions between

resistant oat plants and crown-rust isolates, two BCiFj populations were generated from RI

lines and two parental lines. From these studies, two loci were identified, rdh, a locus

controlling resistance £{ependent ^persensitive lesion development, is recessive and

responsible for the hypersensitive lesion development induced by crown-rust isolate 276.

Rih, a locus controlling Resistance independent ^persensitive lesion development, is dominant and controls the hypersensitive lesion development induced by crown-rust isolate

290.

Biochemical, molecular markers are associated with the necrosis induced by avirulent pathogens (reviewed by Dixon and Lamb, 1990). One of these important markers of resistant response is biosynthesis and deposition of autofluorescence materials, the secondary metabolites in and around death cells (Koga et al. 1980; Koga et al. 1988). They are thought to contribute to limiting pathogen growth and are nearly always associated with plant cells undergoing pathogen-induced hypersensitive cell death (Hahlbrock and Scheel, 1987; Keogh et al. 1980; Koga et al 19881; Woods 1988). To determine whether cell death was involved in fungus-induced hypersensitive lesion development, calcofluor staining (Rohringer et al.

1977) was used to measure the autofluorescence of necrotic host cells at the sites of fungal infection. Two hypersensitive lesion phenotypes were microscopically compared and a susceptible parent was used as a control. These observations indicated that cell death was involved in only resistant plants with hypersensitive lesion development but not in resistant plants without this phenotype or the susceptible plants. The segregation between the resistance phenotype and hypersensitive-lesion-development and its related localized cell death, therefore, suggested that localized cell death is not essential for the resistance. 135

Materials and Methods

Plant materials

The diploid Avena population was generated by crossing A. strigosa (C.I. 3815) with A. wiestii (C,I. 1994) (Rayapati et al. 1994a). A. strigosa is resistant to 40 isolates of P. coronata in the ISU collection whereas A. wiestii is susceptible to the same isolates (Wise and Gobelman-Wemer 1993). One hundred F8.9 RI lines generated by single seed descent from the original F2 mapping population were kindly provided by Dr. M. Lee (Iowa State

University).

BC1F2 populations

To study inheritance of hypersensitive lesion development induced by crown-rust isolate

276, RI A8059 was backcrossed to the resistant parent C.I. 3815. Backcross F, hybrids were selfed to generate a backcrossed F2 (BC1F2) population. The parental line C.I. 3815 was highly resistant and elicited hypersensitive lesion development in response to isolate 276, whereas A8059 was moderately resistant and did not elicit hypersensitive lesion development. To study the inheritance of hypersensitive lesion development induced by crown rust isolate 290, a BC1F2 population was generated from crossing of RI A8073 to C.I.

1994. A8073 was resistant and elicited lesion development to isolate 290, whereas the parent line C.I. 1994 was susceptible and did not elicit lesion development.

Rust inoculations

Two isolates from the ISU collection, 290 and 276, were used to inoculate the recombinant inbred population, BCiF, hybrids and BC1F2 populations. Inoculation and incubation were carried out under our standard conditions (Bush et al. 1994). To measure the concentration of urediospore suspension used in the inoculation, 10 |xl of the urediospore- suspension was placed on a hemacytometer and covered with a cover glass. The number of urediospores was counted for 10 random 0.2mm x 0.2nim x 0.1mm grids under a light 136

microscope. The concentration of the urediospore suspension was calculated as follows; the

total number of urediospores for 10 grids / (10 squares) x 0.2 x 0.2 x 0.1 x (lO'^ml). The

resulting urediospore concentration was 1.2 x 10' urediospores/ml.

To determine the density of urediospores that landed on the leaf surface, glass slides were

held among the seedlings sprayed with during the inoculation. A microscopic field of 0.455

mm in diameter (0.1626 mm^) was counted for the number of urediospores. 10 random fields

were counted. The resulting urediospore density was 3056 urediospores/cm^.

Final disease development was assessed at 7 days after inoculation. On a 0 to 4 scale

using the method of Murphy (1935), infection tyjje reactions (ITR) of 0 or 1 are considered resistant and 3 or 4 susceptible.

Histochemistry and Microscopy

To observe plant defense responses under hypersensitive-lesion-development, non- hypersensitive-lesion-development and susceptible phenotypes at a cellular level, leaf samples of C.I. 3815, A8013, A8087, A8012, and C.I. 1994 were harvested from fungus- infected plants. Hypersensitive lesions were developed and fully recognizable macroscopically in these plants (66 hours after inoculation with isolate 276). C.I. 3815,

A8013, A8012 are resistant and elicit hypersensitive lesion development while A8087, and

A8016 are resistant but do not elicit hypersensitive lesion development. C.I. 1994 are susceptible.

Leaves for autofluorescence examination were cleared and then stained to highlight either the fungal hyphae or necrotic host cell (Rohringer et al. 1977). First, the leaf samples were boiled for 1.5 minutes in lactophenol/ethanol (1:2, v/v) and stored overnight in this mixture at room temperature. They were then washed twice for 15 minutes each with 50% ethanol, twice for 15 minutes each in 0.05 M NaOH, three times with water, and placed in 0.1 M

Tris/HCI buffer, pH 8.5 for 30 minutes. The cleared leaf samples were then stained for 5 137

minutes with a 0.1% solution of cellufluor (used as substitute for calcofluor) (Polysciences,

INC. Warrington, PA) in this buffer. This was followed by washing for three times with

water (10 minutes each) and once with 25% aqueous glycerol (30 minutes). The samples

were mounted in 50% glycerol and examined with Zleiss Research Microscope equipped for

fluorescence microscopy with transmitted light to examine host necrotic cell

autofluorescence and fungal development.

Results

Diverse responses are detected on the RI by inoculation with multiple isolates

The Pea resistance cluster is composed of 5 unique genes that provide multiple

specificities to the oat crown-rust pathogen, P. coronata (Yu and Wise 1999). To study the

specific interactions between the resistance genes and crown-rust isolates, hypersensitive lesion development of seven RI lines and parental lines were investigated in response to infection by six diagnostic crown-rust isolates (Table 1). Crown-rust isolates 290, 263 and

PC62 invoked hypersensitive lesion development on C.I. 3815 and all resistant RI lines. In contrast, crown-rust isolate 276 invoked hypersensitive lesion development only on C.I.

3815, but not on any of resistant RI lines. Crown rust isolates 202 and PC54 did not stimulate any lesion development either on C.I. 3815 or on any resistant RI line. RI lines

A8098 and A9096, although susceptible, still exhibited hypersensitive lesion development to the infection of crown rust isolates 290 and PC62 respectively. These results suggested that hypersensitive lesion development is not always coupled with resistance.

Hypersensitive lesion development is mediated by a unique gene in the C.I. 3815 x C.I.

1994 RI population

To study the inheritance of hypersensitive lesion development induced by isolate 276, the

Fg.9 A. strigosa (C.I. 3815) x A. wiestii (C.1.1994) RI population was analyzed for Table 1. Infection types data of parents and seven RI lines from Avena strigosa (C.I. 3815) x A. wiestii (C.I. 1994) to six isolates Isolate C J. 3815 C J. 1994 A8028 A8060 A8073 A8076 A8085 A8098 A8096 P54 1* 4 2 2 2 2 2 2 2

lHtD« iBSS.

' On a 0 to 4 scale using the method of Murphy (1935). Infection type reaction (ITR) of 0,1 or 2 is considered resistant. ITR of 3 or 4 is considered susceptible. For each line, there were at least three seedlings planted and inoculated. Each result was repeated at least three times. b HLD represents hypersensitive lesion development 139

Table 2. The phenotype data of hypersensitive lesion development in the oat Fg.9 RI population (C.1.3815 x CI. 1994) in response to inoculation of isolate 276.

Parental line F,.,RI population X test

Segregation of HLD among X^-value CJ. 3815' C J. 1994 resistant RI lines (1:1) P-value Resistant Susceptible HLD Non-HLD HLD" Non-HLD 23 28 0.49 0.3-0.5

• Four seedlings were evaluated of each parental line. Infection type reaction (ITR) of 0, 1, or 2 is considered resistant. ITR of 3 or 4 is considered susceptible. '' HLD indicates the presence of hypersensitive lesion development and Non-HLD indicates the absence of hypersensitive lesion development. segregation of hypersensitive lesion development. Lesion development occurred in some resistant plants but not in the others (Table 2). Twenty-three resistant RI lines expressed hypersensitive lesion development phenotypes while twenty-eight did not. These results fit to a 1:1 (hypersensitive lesion development: non-hypersensitive lesion development) ratio, expected for the inheritance of single genes in the RI population. This result indicated that

ITR and the hypersensitive lesion development can segregate independently in the RI population.

A recessive, isolate-specific gene, rdh, mediates the expression of hypersensitive lesion development induced by isolate 276

To further characterize the gene mediating hypersensitive lesion development, RI A8059 was backcrossed to parental line C.I. 3815. RI A8059 is moderately resistant to isolate 276 and lack of lesion development in response to infection of isolate 276. As shown in Table 3, the Fi hybrid of A8059 x C.I. 3815 was resistant and did not exhibit lesion development. All progeny in the A8059 x C.I. 3815 BCiFj population conferred a variable level of resistance to isolate 276, suggesting that both C.I. 3815 and RI A8059 contain resistance genes specific to 140

Table 3. The phenotype data of hypersensitive lesion development in diploid oat BCiFj A8059 X C.I. 3815 population in response to inoculation of isolate 276.

Parental line F1 hybrid BCjFj population X test

A8059X Segregation X^-value C J. 3815* A8059 C J. 3815 of resistance and HLD (1:3) P-value

Resistant Resistant Resistant Resistant Resistant HLD Non-HLD HLD*" Non-HLD Non-HLD 25 88 0.49 0.3-0.5

* Four seedlings were evaluated of each parental line. Infection type reaction (ITR) of 0, 1, or 2 is considered resistant. ITR of 3 or 4 is considered susceptible. HLD indicates presence of hypersensitive lesion development and Non-HLD indicates absence of hypersensitive lesion development. isolate 276. Segregation of progeny in the population with hypersensitive lesion development to those without was a good fit to 1:3, hence, indicating that a recessive gene mediated hypersensitive lesion development A dominant hypersensitive-Iesion-deveiopment gene, Rih, is detected in the A8073 x C.l.

1994 BC1F2 population in response to the infection by isolate 290

The inheritance of hypersensitive lesion development was also analyzed by inoculating the A8073 x C.I. 1994 BCiFj hybrid and A8073 x C.I. 1994 BC1F2 population with isolate

290. As shown in Table 4, RI A8073 displayed a high level of resistance in addition to hypersensitive lesion development similar to C.I. 3815. The F, hybrid plants exhibited both a resistant response and hypersensitive lesion development, indicating that dominant genes controlled the resistance specificity and hypersensitive lesion development respectively.

Among three hundred and fifteen plants in the BC1F2 population, two hundred and twenty- one plants developed lesions while 94 plants did not. Segregation of progeny with hypersensitive lesion development to those without was a good fit to 3:1, indicating that a Table 4. Hypersensitive lesion development in the A8073 x C.I. 1994 BCiFjpopulation in response to inoculation of isolate

290

Parental line Fihybrid BCiFj population X^test for HLD

A8073X X^-vahie A8073' CJ.1994 CJ.1994 Segregation of HLD Segregation of ITR' (3:1) P-value

Resistant Susceptible Resistant HLD Non-HLD Resistant Susceptible

HLD'' non-HLD HLD 221 94 225 90 3.68 0.05-0.10

' Four seedlings were evaluated of each parental line. Infection type reaction (ITR) of 0,1, or 2 is considered resistant. ITR of 3 or 4 is considered

susceptible.

'' HLD indicates presence of hypersensitive lesion development and Non-HLD indicates absence of hypersensitive lesion development.

2 ' X test indicated that segregation of ITR was well fit to the inheritance of a dominant gene. 142 single dominant gene mediating the hypersensitive lesion development. Table 5 lists the segregation data between hypersensitive lesion development phenotypes and resistance. Of the 221 lines that displayed hypersensitive lesion development, 215 were resistant plants and

6 were susceptible. Of the 94 plants that did not display hypersensitive lesion development,

10 were resistant and 84 were susceptible. This result further indicated that hypersensitive lesion development was genetically controlled by an additional factor.

Linkage analysis indicates that the two hypersensitive lesion development genes, rdh and Rih, are located on different diploid linkage groups

To determine the relationship between the two hypersensitive-lesion-development loci and other resistance related genes, the map location of these loci were investigated.

Hypersensitive lesion development was elicited in only the resistant RI lines in the A. strigosa (C.I. 3815) x A. wiestii (C.I. 1994) recombinant inbred population in response to isolate 276. Hence, the genotypes for 51 resistant RI lines at the rdh locus can be determined. RI lines that exhibit hypersensitive-lesion-development phenotypes were assumed to be homozygous for the recessive allele and RI lines that correspond for non- lesion-development genotypes were assumed to be homozygous for the dominant allele.

These RI data were integrated into the Map Manager data file of our newly established diploid Avena map (Yu and Wise 1999) and rdh was positioned on linkage group A between

Bare-E12-9a and Bare-E14-la, two Bare-based S-SAP markers (Yu and Wise 1999). The segregation analysis of Rih in the RI A8073 x C.I. 1994 BC1F2 population indicated thati?//i is tightly linked with resistance gene Pc82 which is located within the Pea resistance gene cluster on diploid Avena linkage group B (Yu and Wise 1999). As indicated in Table 5, among the 315 plants inoculated with isolate 290, 6 susceptible plants did elicited hypersensitive lesion development whereas 10 resistant plants did not. A Chi-square test indicated that Pc82 and Rih were not genetically independent. The genetic distance between 143 the two loci was estimated based on the segregation within the BCiFj population. If the recombination ratio is designated as r, then the ratio for homozygous recessive plant lines

(non-lesion-development and susceptible) in the BCiFj population would be 1/4 (1-r)^. This ratio is 84/315. The recombination ratio between the two loci can be calculated by solving equation of 1/4 (1-r)^ = 84/315. Rih is about 3.3 cM away from the Pc82 resistance gene and, therefore, located within the Pea resistance gene cluster but its accurate position within the

Pea cluster is yet to be determined.

The relationship between lesion development and localized cell death

Autofluorescence is a hallmark of plant cells undergoing fungus-induced hypersensitive cell death. To determine the relationship between localized cell death and hypersensitive lesion development in response to crown-rust isolates, the accumulation of autofluorescence was assayed by calcofluor staining. Fig. 1 illustrates the interaction between fungal hyphae and plant host cells. As indicated in panel A, the hypersensitive lesion development was accompanied with plant cells undergoing hypersensitive cell death. Necrotic host cells fluoresced a bright, orange-yellow while hyphae of crown rust fungus was bright blue and unaffected plant cells displayed a green fluorescence. No necrotic host cells were observed in resistant, non-hypersensitive lesion development (panel B) or susceptible plants (panel C).

To determine the rate of host cell death, both the number of cell death sites and the number of fungal growth sites within the mesophyll cells were counted on leaf samples under a microscope. As indicated in Table 6, the hypersensitive lesion development was coupled with a high ratio of cell death. Among 865.1 fungus growth sites/cm^, 1036 clusters of dead cells were observed in C.I. 3815 with a cell death rate of 1.198. In contrast, there was a cell death rate of only 0.026 in A8016 and 0.012 in C.I. 1994. The germination of urediospores appears to be inhibited on the leaf surface of the plants with LCD phenotype. Of 3086 urediospores /cm^ landed on the leaf surface at the time of inoculation, there was there was a Fig. 1. Response of hypersensitive-lesion-development, non-hypersensitive-lesion-

development, and susceptible plants to fungal pathogen. P. coronata isolate 276 was used to

infect hypersensitive-lesion-development (C.I. 3815) (A), non-hypersensitive-lesion-

development (A8016) (B), and susceptible plants (C.I. 1994) (C). In panel A,

autofluorescence was shown to accumulate in clusters of plant host cells (bright, orange-

yellow) adjacent to fungal hyphae (bright blue). Healthy plant host cells was at background

(bight green). In contrast, no autofluorescence was accumulated in the non-lesion- development plants (B) and in the susceptible plants (C). rdh is a recessive gene that

mediates hypersensitive lesion development induced by isolate 276. Pc82 is a resistance

gene that confers resistance specificity to isolates 276. Genotype for C.I. 3815 is rdhlrdh,

Pc82/Pc82, genotype for C.I. 1994 is Rdh/Rdh,pc82lpc82 and genotype for A8016 is

RdhlRdh,Pc82IPc82. (aiH) si.8e TO V

SPl Table 5. The phenotype data of hypersensitive lesion development in diploid oat BCiFj A8073 x C.I. 1994 population in response to inoculation of isolate 290

Parental line F, hybrid BCiFj population %^est

A8073X X^-value A8073* C J. 1994 C J. 1994 Cosegregation of resistance and HLD (9:3:3:1) Resistant Susceptible Resistant Susceptible Resistant HLD Non-HLD HLD Non-HLD HLD" non-HLD HLD 215 10 6 84 306.3 <0.001*

" Four seedlings were evaluated of each parental line. Infection type reaction (ITR) of 0,1, and 2 is considered resistant. ITR of 3 and 4 is considered susceptible. HLD indicates presence of hypersensitive lesion development and Non-HLD indicates absence of hypersensitive lesion development. Table 6. Frequency of cell-death sites on plants with different HLD phenotypes in response to infection of isolate 276.

Germinated Number of sites Number of sites urediospores Germination rate of fungal growth of ceU death Cell death rate Plant line Phenotype* (NlVcm^ (Nl/3086') sites (N2'')/cm^ (N3')/cm^ (N3/N2) C.I. 3815 R&HLD 880.6 0.288 865.1 1036 1.198 C.1.1994 S & Non-HLD 1146.9 0.372 1078.5 13.39 0.012 A8016 R& Non-HLD 995.4 0.323 1078.35 27.65 0.026

'R represents resistance response and S represents susceptible. HLD indicates presence of hypersensitive lesion development and non-HLD indicates absence of hypersensitive lesion development. Leaf samples were harvested 66 hours after inoculation. •"Nl is the number of germinated urediospores observed on the leaf surface at the time of microscopic observation. 3086 (urediospores/cm^) is the urediospores density on the leaf surface at the time of inoculation. ''N2 is the number of sites counted where the growth of fungal hyphae was successfully established among mesophyll cells. ®N3 is the number of sites where cell death occurred among the N2. 148

germination rate of 0.32 in RI A8016 but only 0.288 in C.I. 3815 whereas the susceptible

parent C.I. 1994 had a rate of 0.372.

Discussion

In plants, a highly specialized form of recognition is called racexultivar specificity and

the interaction is govemed by gene-for-gene interaction (Flor 1971). The response triggered

by the interaction is complex and comprises a battery of molecular, biochemical, cellular and

physiological responses. Localized cell death at infection sites is one of the most significant components involved in the plant defense response (Lawton 1997).

In diploid Avena, hypersensitive lesion development at the site of fungal infection is often a typical characteristic of resistance to the crown-rust pathogen. In this report, a novel phenomenon was observed in that the resistance phenotype could be genetically separated from hypersensitive lesion development in response to infection by crown-rust isolate 276.

Among fifty-one resistant RI lines, 23 exhibited hypersensitive lesion development phenotypes while 28 did not, suggesting that additional genetic factors control this phenotype. To further characterize these factors, segregation of hypersensitive lesion development was analyzed in response to infection by isolates 276 and 290 in the A8059 x

C.I. 3815 and A8073 x C.I. 1994 BC1F2 populations, respectively. The rdh mdRih loci were identified. The observation that rdh segregated independently of Pc82 and that recombinants were detected between Rih and Pc82 indicated that additional factors mediated the hypersensitive lesion development. In contrast, hypersensitive lesion development was never elicited in response to inoculation of crown-rust isolates 202 and PC54. This observation further supported the assumption that that the hypersensitive lesion development was controlled by the separate genetic factors from the primary resistance gene. 149

Autofluorescence is one of the most important biochemical and molecular markers of plant resistance response. This marker displays host cells that undergo hypersensitive cell death. Calcofluor staining was used to efficiently assay the accumulation of autofluorescence. The interactions of plant host cells and fungal hyphae on plants of two different lesion phenotypes were analyzed. These results indicated that hypersensitive lesion development was accompanied with a high ratio of number of occurrences of necrotic cells to the number of fungal growth sites observed. In contrast, the non-hypersensitive-lesicn- development plants and the susceptible plants had much lower ratio of cell death. This observation indicated that hypersensitive lesion development was related to hypersensitive cell death.

Compared to the genes responsible for the lesion mimics, the hypersensitive-lesion- development controlling loci, rdh and Rih, showed specific interactions between oat host lines and crown rust isolates. Therefore, these loci and the relationship with crown-rust isolates are more likely the result of co-evolution between the plant host and the pathogen.

In contrast, the alleles responsible for tiie lesion mimics may be derived either from the resistance gene mutation (Walbot et al. 1983; Johal et al. 1995) or from the mutation of the genes in controlling cell development (Jackobek and Lindgren 1993).

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CHAPTER 6. GENERAL CONCLUSION

Conclusions

Comparative genetics mapping has shown a high level of conservation of gene content

and gene orders within genomes. This research has established a foundation for cross-

species information exchange. In addition, comparative mapping focused on a particular

gene provides information to determine orthologous relationship among genes in different

species. In Avena, Hordeum, and Zea mays, thirty-two DNA probes from homoeologous group 1 chromosomes of the Gramineae were mapped. The resulted map showed conserved linkage relationships between these markers and several resistance genes (Yu et al. 1996).

These resistance loci included the Pea crown rust resistance cluster in diploid oat, the R203 crown rust resistance locus in hexaploid oat, the Mia powdery mildew resistance cluster in barley. Data from the maize populations were integrated into the Brookhaven maize RFLP map by Dr. B. Burr at Brookhaven National Laboratory. The results showed that six resistance genes, rp3,wsml,wsm2, mdml, ht2, and htnl previously mapped on maize chromosomes (Coe 1993; Simcox and Bennetzen 1993; McMuUen et al. 1994; Simcox et al.

1995) were located within the three homoeologous linkage groups. The highly conserved linkage between resistance genes and heterologous markers suggested that these resistance genes may be orthologs. If it could be further verified, this result may implicate that different plant species may utilize similar mechanisms against disease pathogens. The homoeologous fragments in diploid oat and barley were basically duplicated in maize, further supporting that maize is descended from an ancient tetraploid species (Ahn et al. 1993).

Disease resistance genes provide an effective way in preventing disease. They tend to be clustered in the genomes (Wise and Ellingboe, 1985; Hulbert and Bennetzen, 1991; Islam and Shepherd, 1991; Gorg et al. 1993). In diploid Avena, Pea, a crown-rust resistance locus. 156

confer resistance to nine crown rust isolates of P. coronata (Rayapati et al. 1994a, 1994b;

Wise et al. 1996). Recombination breakpoint analysis of seven BQFj populations identified

five tightly linked genes within the cluster; Pc81, Pc82, Pc83, Pc84, and Pc85. They

controlled six crown rust resistance specificities; R54, R276/R290, R263, R62 and R202

respectively. This result provided evidence that Pea is a resistance gene family. AFLP

based bulk analysis identified three markers, Agx4, Agx7, and Agx9. They are closely linked

to Pea. Agx4 cosegregates with the Pe85 resistance gene. These markers will be very useful

in research of evolution of resistance specificity at the locus and in marker assisted resistant

breeding.

Genetic linkage map of high resolution establishes a foundation for various genetic

researches such as gene targeting, positional cloning (Tanksley et al. 1995). Using AFLP

and retrotransposon-based S-SAP, a saturated diploid Avena map was established in A.

strigosa (C.I. 3815) x A. wiestii (C.I. 1994) RI population. This map includes 369 AFLP and

80 S-SAP markers, six crown-rust resistance genes, a cluster of resistance-gene analogs, and

one morphological marker, and is anchored by 45 grass-genome RFLP markers. The linkage

map showed consensus with diploid Avena map from A. atlantica x A. hirtula population

(Van Deynze et al. 1995b). Mapping Bare-l-like retrotransposon indicated that long terminal

repeat (LTR) of barley Bare-l-like retrotransposon has a considerable degree of

heterogeneity and widespread distribution on diploid Avena genome. The RI population has

been widely used in the Avena genetic researches (R. Wise, personal communication).

Therefore, this saturated map will be the useful framework for these researches.

Localized cell death is a nearly ubiquitous feature of incompatible interaction of plants

and microbe pathogens. It is assumed that cell death could prevent spreading of pathogen

(Heath 1980; Dangl 1995; Staskawicz et al. 1995). Two new loci that control hypersensitive cell death were identified. Both loci segregated independently from the Pc82 crown-rust 157

resistance gene, rdh, a locus controlling resistance dependent hypersensitive response, is

recessive and responsible for lesion development induced by crown-rust isolate 276. Rih, a

locus controlling Resistance independent iiypersensitive response, is dominant and controls

lesion development by crown-rust isolate 290. Microscopic observation indicated that cell

death was involved in the interaction between plant host cells and crown-rust fungus in plants

with hypersensitive lesion development. Segregation between lesion development and

resistance gene suggested that hypersensitive lesion development and its related cell death

may not be essential for plant resistance. Further characterization of these genes will help us to understand mechanisms of cell death and facilitate possible utilization of cell death genes in plant resistant breeding.

Recommendations for Future Research

Plant resistance genes confer resistance to diverse pathogens but encode proteins that share highly conserved amino acid motifs (Michelmore 1995; Lawton 1997; Michelmore

1998). It is interesting to ask questions such as: are these genes evolved from the same resource or are they orthologs? If they were orthologs, they would share mechanisms in conferring resistance against plant pathogens. Although conserved linkage relationships between these markers and some resistance genes were found in Avena, Hordeum, and Zea mays, enough evidence has not yet been obtained to be able to answer the above questions.

Two experiments can be designed to obtain the much-needed information. First, fine genetic syntenic maps can be established for each of the resistance gene regions using additional set of heterologous DNA probes. Second, when the resistance gene tightly linked or cosegregated DNA markers were available, BAC or YAC clones could be screened from corresponding genomic libraries and microsyntenic relationships could be established. In both cases, clue about the relationship of these resistance genes can be obtained. 158

As we know, one of the parents of the RI population, A. strigosa, is resistant to 40

isolates of P. coronata in the ISU collection whereas A. wiestii is susceptible to the same

isolates (Wise and Gobelman-Wemer, 1993). Therefore, the RI population and possibly the

BCiFj populations can be used to differentiate and map additional resistance specificity and

their corresponding genes. It would not be surprised to find that more resistance genes be

positioned in the Pea resistance cluster.

It is also interesting to know whether retrotransposons are still active in the Avena

genome. The result obtained from our research may suggest that these elements may be

active in the Avena genome but result is far from conclusive. The activity of retrotransposon

in the Avena genome can be studied by tracing back the early generation of the RI population

using retrotransposon-based S-SAP technique. Therefore, effect on genome evolution and

gene regulation of activity of retrotransposon could possibly be determined.

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Phytopathology 86:340-346.

Yu, G. X., Bush, A. L., and Wise, R. P. 1996. Comparative mapping of homologous group 1 regions and genes for resistance to obligate biotrophs in Avena, Hordeum, and Zea mays.

Genome 39:155-164. 161

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my major professor. Dr. Roger Wise.

He is a good mentor who always guided me in my research. He is a good English teacher

who taught me how to be a good speaker as well as to be a good writer, especially. His support helped me satisfy my dream to be an excellent geneticist and, yet, to be a

bioinformatic scientist.

I also appreciated the willingness of Dr. Patrick Schnable, Randy Shoemaker, Steven

Rodermel, and Edward Braun to serve as my member of my POS committee and to provide comments and advice on my research project. In addition, I would like to acknowledge all my colleagues in the lab who always give me help whenever needed. Especially, I would like to thank Mrs Karin S. Gobelman-Wemer, our lab technician, who always gave me technique and personal support whenever I was in trouble. I would like to express my appreciation to Dr. Aria L. Bush and Dr. Richard A. DeScenzo, one-time research associates in our lab. They gave me their precious support and understanding. Their friendship helped me overcome the hardest time during my research.

I would like to use the chance here to express my thanks to my family, my wife Gu Xie and my daughter Michelle Yu. Their support and encouragement are the main force with which I have been able to complete this degree.