ABSTRACT

MAXWELL, JUDD JOSEPH. Genetic Characterization and Mapping of Wheat Powdery

Mildew Resistance Genes from Different Wheat Germplasm Sources. (Under the direction of

J. Paul Murphy).

Powdery mildew caused by Blumeria graminis f. sp. tritici is a major economic

disease in wheat (Triticum aestivum) in cool and humid, or maritime environments. Grain

yield loss can reach 48% in susceptible cultivars under severe epidemics. Race-specific host

resistance was identified as the most effective, consistent, and economic method of powdery

mildew control. However, because of the constant virulence shifts and recombination among

powdery mildew isolates, most race-specific resistance genes are ephemeral and are

overcome a few years after wide deployment. Incorporation of new and novel resistance

genes is imperative to maintain effective control in new wheat cultivars. The wheat

germplasm lines NC96BGTD1, NC97BGTAB10, NC06BGTAG12, and NC06BGTAG13

exhibit different virulence spectra and a high level of powdery mildew resistance in the

southeastern United States. The objectives of this study were to characterize the inheritance

of the powdery mildew resistance and identify simple sequence repeat (SSR) markers linked

to the resistance genes in each line. The NC96BGTD1 and NC97BGTAB10 lines were

crossed to the cultivar Saluda and F2:3 families were developed for field and greenhouse

evaluations. The NC06BGTAG12 and NC06BGTAG13 lines were crossed to the susceptible

cultivar Jagger and F2:3 families were developed for greenhouse evaluation. SSR markers

were used to develop linkage maps to localize the genes to their respective chromosomes in

each population. Phenotypic segregation among the F2:3 families indicated monogenic

control of the resistance gene in each of the four populations.

The resistance in NC96BGTD1 was located to the short arm of chromosome 7D,

flanked by the SSR markers Xwmc635 and Xcfd41 at a genetic distance of 5.3 cM distal and

20 cM proximal, respectively. Because seed of Pm19 was not available in a timely fashion,

we were unable to determine if the resistance gene in NC96BGTD1 was a novel resistance

gene or an allele of the Pm19 locus which is also located on chromosome 7. The gene in

NC96BGTD1 was given the temporary designation MlNCD1.

The resistance gene in NC97BGTAB10 was located to the terminal tip of

chromosome 2BL, with the SSR marker Xwmc445 mapping 7 cM proximal to the gene.

Again, because seed of MlZec1 was not available in a timely manner, we were unable to

determine the relationship between the gene in NC97BGTAB10 and the powdery mildew

gene MlZec1, which is also located on chromosome 2BL. The gene in NC97BGTAB10 was

given the temporary designation MlAB10

The resistance genes in NC06BGTAG12 and NC06BGTAG13 mapped to the same

region of chromosome 7AL and an allelism test indicated that the genes were likely allelic.

Furthermore, the genes mapped to the same region where the Pm1 locus resides, but a

detached leaf test suggested that the genes were different from each other as well as all five

of the alleles at the Pm1 locus. Linkage mapping showed that the resistance genes were

flanked by the SSR markers Xwmc273 and Xwmc346 by a distance of 7.2 cM distal and 2.4

cM proximal respectively, in NC06BGTAG12 and 8.3 cM distal and 6.6 cM proximal

respectively, in NC06BGTAG13. We were unable to determine the linkage or allelic

relationship of the resistance genes in NC06BGTAG12 and NC06BGTAG13 with the Pm1

locus at this time. Thus these genes in NC06BGTAG12 and NC06BGTAG13 were given the

temporary designation MlAG12 and MlAG13, respectively.

Genetic Characterization and Mapping of Wheat Powdery Mildew Resistance Genes from Different Wheat Germplasm Sources

by Judd Joseph Maxwell

A dissertation submitted to the Graduate Faculty of North Carolina State University

In partial fulfillment of the Requirements for the degree of

Doctor of Philosophy

Crop Science

Raleigh, North Carolina

2008

APPROVED BY:

_______________________________ ______________________________ James Holland Gina Brown-Guedira ________________________________ ________________________________ David Marshall Christina Cowger ________________________________ J. Paul Murphy Committee Chair

ii

DEDICATION

I would like to dedicate my dissertation to my grandparents, Joseph and Nancy

Zearing. Some of my earliest memories are gardening with my grandpa, and going out in the

field to talk with the framers that work the land we owned. They both had a profound

influence on my life which gave me the aspiration and the spiritual support to get where I am

today.

iii

BIOGRAPHY

Judd Joseph Maxwell was born and raised in the small Midwestern town of Princeton,

IL. After graduating from Princeton High School, Judd went to Colorado State University

for his undergraduate studies and receiving his Bachelor of Science degree in 2002. He

continued on at Colorado State for his masters studies, where he worked with Dr. Mark Brick

studying white mold resistance in common bean. Promptly after receiving his Masters of

Science in plant breeding and genetics in 2005, he relocated to Raleigh, NC to work on his

Ph.D. with Dr. J. Paul Murphy in the small grains program at North Carolina State

University. After the completion of his Ph.D. degree, Judd will work for Monsanto as a Line

development corn breeder in Independence, IA.

iv

ACKNOWLEDGEMENTS

I would first like to thank my committee for their input, suggestions, and

encouragement throughout my time here at N.C. State. I don’t think I would have made

without all your help. A special thanks to Paul Murphy for taking me the back ways to the

fields and exposing me all of the great North Carolina cuisine.

I am grateful to my colleagues in the small grains program who aid me with help in

planting, lab techniques, greenhouse work, and crossing. Jeanette Lyerly, Rene Navarro, Dr.

David Wooten, Peter Maloney, Sydney Jarrett, you guys were the best helpers I could ask

for. I am also grateful to Ryan Parks, Abbey Sutton, and Lynda Whitcher for all their help in

the pathology lab for increasing isolates and helping set up my differential tests.

I want to thanks Dr. Leandro Perugini and Jared Smith for their friendship and

support.

I am very thankful and appreciative to my family for their continual support and

encouragement throughout my college career. I know it hasn’t been easy and I couldn’t have

done it without you. I would also like to give thanks to my dog Karma for her patience and

always reminding me to take a break and go for a walk.

Lastly I would like to thank Jen Petzold for her support the past couple of years; you

have made this a lot more fun and less painful.

v

TABLE OF CONTENTS

LIST OF TABLES ------------------------------------------------------------------------------- vii LIST OF FIGURES ----------------------------------------------------------------------------- viii

CHAPTER I. LITERATURE REVIEW ---------------------------------------------------------- 1

Introduction --------------------------------------------------------------------------------- 2 Wheat ---------------------------------------------------------------------------------------- 3 Wheat powdery mildew ------------------------------------------------------------------- 4 Powdery mildew resistance in wheat ---------------------------------------------------- 7 Sources and breeding for powdery mildew resistance --------------------------------- 8 Race-specific resistance ------------------------------------------------------------------- 9 Adult plant resistance -------------------------------------------------------------------- 11 Gene postulation -------------------------------------------------------------------------- 12 Genetic Localization --------------------------------------------------------------------- 13 Genetic and molecular mapping -------------------------------------------------------- 15 RFLP analysis ----------------------------------------------------------------------------- 16 RAPD analysis ---------------------------------------------------------------------------- 17 AFLP analysis----------------------------------------------------------------------------- 17 SSR analysis ------------------------------------------------------------------------------ 18 Other marker systems -------------------------------------------------------------------- 19 Marker assisted selection (MAS) ------------------------------------------------------- 20 Powdery mildew resistance breeding at North Carolina State University -------- 22 References --------------------------------------------------------------------------------- 25

CHAPTER II. MlNCD1: A Novel Aegilops tauschii-Derived Powdery Mildew Resistance Gene in Common Wheat ------------------------------------------------------------------------- 45

Abstract ------------------------------------------------------------------------------------ 46 Introduction ------------------------------------------------------------------------------- 47 Materials and Methods ------------------------------------------------------------------- 50

Plant material --------------------------------------------------------------------- 50 Greenhouse evaluation ---------------------------------------------------------- 50 Field evaluation ------------------------------------------------------------------ 52 Detached leaf test ---------------------------------------------------------------- 54 Linkage analysis ----------------------------------------------------------------- 54

Results ------------------------------------------------------------------------------------- 56 Greenhouse and field evaluations --------------------------------------------- 56 Marker analysis ------------------------------------------------------------------ 57

Discussion --------------------------------------------------------------------------------- 60 Acknowledgments ------------------------------------------------------------------------ 63

vi

References --------------------------------------------------------------------------------- 64

CHAPTER III. MlAB10: A Triticum turgidum-Derived Powdery Mildew Resistance Gene Identified in Common Wheat -------------------------------------------------------------------- 73

Abstract ------------------------------------------------------------------------------------ 74 Introduction ------------------------------------------------------------------------------- 75 Materials and Methods ------------------------------------------------------------------- 78

Plant material --------------------------------------------------------------------- 78 Greenhouse evaluation ---------------------------------------------------------- 79 Field evaluation ----------------------------------------------------------------- 80 Marker analysis ------------------------------------------------------------------ 81

Results ------------------------------------------------------------------------------------- 84 Greenhouse and field evaluations --------------------------------------------- 84 Marker analysis ------------------------------------------------------------------ 84

Discussion --------------------------------------------------------------------------------- 87 References --------------------------------------------------------------------------------- 90

CHAPTER IV. Identification and Genetic Mapping of Two Triticum timopheevii-Derived Powdery Mildew Resistance Genes in Common Wheat ------------------------------------ 100

Abstract ----------------------------------------------------------------------------------- 101 Introduction ------------------------------------------------------------------------------ 103 Materials and Methods ------------------------------------------------------------------ 106

Plant materials ------------------------------------------------------------------ 106 Phenotypic evaluation ---------------------------------------------------------- 107 Marker analysis ----------------------------------------------------------------- 109

Results ------------------------------------------------------------------------------------ 112 Inheritance of powdery mildew resistance --------------------------------- 112 Molecular mapping ------------------------------------------------------------- 113 Detached leaf test --------------------------------------------------------------- 114

Discussion -------------------------------------------------------------------------------- 116 Acknowledgments ----------------------------------------------------------------------- 120 References -------------------------------------------------------------------------------- 121

vii

LIST OF TABLES

Table 1.1: Formally designated powdery milder resistance genes, their chromosomal location, source of the resistance, fist reference for each gene, subsequent PCR based linked molecular markers type(s), and reference for marker(s) -------------------------------------- 39 Table 1.2: Temporary designated wheat powdery mildew resistance genes, their chromosomal location, source, and reference -------------------------------------------------- 42 Table 1.3 NC State wheat powdery mildew resistant germplasm lines, pedigree, source of the resistance, chromosomal location of the resistance gene, and gene desigantation -------- 43 Table 2.1 Segregation ratios for powdery mildew reaction of F2:3 families from the NC-D1 / Saluda wheat population in greenhouse and field evaluations -------------------------------------------------- 68 Table 2.2 Segregation ratios for SSR markers among F2 individuals in the NC-D1/Saluda wheat population ----------------------------------------------------------------------------------------------------- 69 Table 2.3 Detached leaf reaction of NC-D1, XX186, Saluda, and the susceptible check chancellor to five powdery mildew isolates ------------------------------------------------------------------------------ 70 Table 3.1. Segregation ratios for powdery mildew reaction of F2:3 lines from the NC-AB10 / Saluda wheat population evaluated in two replications in both greenhouse and field tests - ------------------------------------------------------------------------------------------------------- 95 Table 3.2 Powdery mildew reactions of differential wheat lines and NC-AB10 under infection with natural inoculum in the field evaluation in 2008 ----------------------------- 96 Table 3.3 Segregation ratios for SSR markers among F2 individuals in the NC-AB10 / Saluda wheat population -------------------------------------------------------------------------- 97 Table 4.1 Segregation ratios for powdery mildew reaction of F2 derived families from the NC-AG12 x Jagger (NC-AG12), NC-AG13 x Jagger (NC-AG13), and NC-AG12 x NC-AG13 crosses ------------------------------------------------------------------------------------- 125 Table 4.2 Segregation ratios for the SSR markers that flank the powdery mildew resistance genes in NC-AG12 and NC-AG13 ------------------------------------------------------------- 126 Table 4.3 Reaction of six genotypes possessing Pm1 alleles, NC-AG12, NC-AG13, Saluda, Jagger, and the susceptible control Chancellor after infection with ten isolates of Blumeria graminis f. sp. tritici (Bgt) ----------------------------------------------------------------------- 127

viii

LIST OF FIGURES

Figure 2.1 Map position of Pm 41 on chromosome 7DS. The gray arrow indicates the direction of the centromere ------------------------------------------------------------------------------------------------ 71 Figure 2.2 Chromosome and deletion bin localization of SSR marker Xgwm635 PCR products observed in Saluda (susceptible allele), NC-D1 (resistant allele), Chinese Spring (CS), Ditelosomic 7Ds (Dt7DS), and the deletion line 7DL-6, but no PCR product was observed in the nullisomic 7D (N7D-T7B), Ditelosomic 7DL (Dt7DL), or deletion stocks 7DS-4, 7DS-1, and 7DS-5 ----------- 72 Figure 3.1 Mapping position of MlAB10 on chromosome 2BL. The gray arrow indicates the direction of the centromere ----------------------------------------------------------------------- 98 Figure 3.2 Gel image of the CS anueploid and deletion stocks used to confirm the location of the linked SSR markers Xgwm526 (A) and Xwmc445 (B), lanes 1-11 represent NC-AB10, Saluda, Chinese Spring, N2BT2D, N2DT2B, Dt2BL, Dt2BS, del 2BL-3, del 2BL-5, del 2BL-10, del 2BL-6, respectively ---------------------------------------------------------------- 99 Figure 4.1 Map positions of the wheat powdery mildew resistance genes in NC-AG12 (A) and NC-AG13 (B) on chromosome 7AL. The gray arrow indicated the direction toward the centormere. Marker names are to the right and the distances between markers in centimorgans (cM) are to the left --------------------------------------------------------------- 128

Chapter I

Literature Review

2

Introduction

Common wheat (Triticum aestivum L.) is one of the most widely produced grain

crops worldwide with an annual production of over 600 million metric tons (Mt) (FAO;

Http://www.fao.org/). This is a forty percent increase from the 250 million Mt produced

in 1965. The increase in production has not come from an expansion in the area sown to

wheat, but rather it has come through the increase of yield per hectare (Curtis 2002). A

one percent per annum yield increase was achieved over the past 20 years using modern

cultivars under irrigated conditions and a 2-3 percent per annum increase under marginal

conditions (Reynolds and Borlaug 2006). Much of the production increase resulted from

technological advances such as better fertilizers, field equipment, pest control, agronomic

practices, and improved genetics (Marshall et al. 2001). Genetic improvement through

yield stability across environments has largely come by the adoption and management of

high-yielding, disease resistant, semi-dwarf wheat cultivars throughout the world,

particularly in developing nations (Curtis 2002).

Fifty three percent of wheat production is in developed countries. Forty-seven

percent is in developing countries, and this is expected to rise to 50 percent by 2030.

Two main elements will contribute to the shift in production and increased demand for

grain production in the years to come: (1) population increase, particularly in the

developing countries, and (2) the increase in caloric intake in most developing countries

(Marathee and Gomez-MacPherson 2001). With the increase in production and demand

3

in the developing nations, a reduction in input cost for wheat production will need to be

achieved for sustainable wheat production. With increased fuel and pesticide costs,

resource poor farmers are relying on genetic resistance to pests and diseases to a greater

degree. For this reason breeders have placed emphasis on breeding for genetic disease

resistance to protect farmers from uncertainty and the environment from the

agrochemical burden (Reynolds and Borlaug 2006).

Wheat

Wheat was domesticated circa 10,000 years ago in the Fertile Crescent arching

from Iran in the east, through the Tigris and Euphrates basins, Syria, central Israel, and to

Jordan in the west (Feldman 2001; Salamini et al. 2002; Mori et al. 1997). Common

wheat is a true breeding allohexaploid (2n = 6x = 42) composed of three genomes, A, B,

and D, which were derived from diploid and tretraploid wheat species. Triticum uratu

Tum. ex Grand. contributed the A genome, Aegilops speltoides Tausch, or an extinct

close relative, most likely contributed the B genome, and Ae. tauschii Coss. contributed

the D genome (Hsam and Zeller 2002; Salamini et al. 2002). Common wheat is the

product of hybridization between the tetraploid wheat T. turgidum ssp. dicoccoides

(Kron. ex Asch. & Graebner) Thell. and the diploid wheat Ae. tauschii, followed by

chromosome doubling (Feldman 2001).

4

Wheat powdery mildew

Powdery mildew, caused by Blumeria graminis (DC.) Speer f. sp. tritici (Em.

Marchal) (syn. Erysiphe graminis f. sp. tritici) is an economically important pathogen

that affects grain yield and end-use quality in wheat. B. graminis is a biotrophic foliar

fungus which seldoms kills its host (Forsstrom 2002). B. graminis is classified as a

higher fungus of the subdivision Ascomycotina in the order Erysiphales. Powdery

mildew is most widespread and severe at temperate latitudes and in coastal areas with

maritime climates, but occurs wherever wheat is produced (Bennett 1984; Hsam and

Zeller 2002). It has been suggested that powdery mildew has become an important

disease in drier and hotter areas because of changes in agricultural practices such as the

increased use of irrigation, genetically uniform semi-dwarf cultivars, and nitrogen

fertilizers (Bennett 1984; Imani et al. 2002). These new agricultural practices produce a

microclimate within the canopy that is conducive to powdery mildew epidemics.

Powdery mildew in the southeastern United States can be found continuously on

winter wheat following emergence in the fall until late spring. It is observed first on the

oldest leaves and progresses upward with plant growth (Bowen et al. 1991). Symptoms

of powdery mildew infections include patches of cottony mycelium and conidia on the

leaf surface (Wiese 1987). B. graminis can complete its life cycle in 10 days under

optimal conditions of temperatures ranging between 15-20oC and high humidity, which

can lead to multiple life cycles throughout the year. Temperatures above 25oC can

5

severely retard the growth of powdery mildew, which causes disease progression to slow

as the growing season progresses into the hotter months (Schafer 1987; Leath and Bowen

1989). Powdery mildew spores are wind dispersed. Thus wind is the main weather

factor that increases the probability of occurrence of a powdery mildew epidemic, while

temperature and humidity have the largest impact on the severity of an epidemic (Te

Beest et al. 2008).

Powdery mildew negatively affects wheat yield and flour quality by altering the

growth habit and metabolism of wheat plants. Early powdery mildew infection of wheat

seedlings stimulates tiller production (Everts and Leath 1992). This increased tiller

production depletes carbohydrate reserves, resulting in fewer tillers surviving until

maturity and fewer kernels forming per tiller (Everts and Leath 1992). Powdery mildew

infections will alter the types of sugars and enzymes produced in the leaves which are

needed for transport of starches to the phloem, thus preventing carbohydrate

accumulation in the seeds (Schulze-Lefert and Panstruga 2003; Wright et al. 1995).

Infections will also cause an increase in transpiration and respiration while lowering net

photosynthesis rates of the infected plants (Shtienberg 1992; Zulu et al. 1991). These

reductions in starch and carbohydrate production and the increase in transpiration have a

major negative impact on kernel weight and kernel size (Bowen et al. 1991). Together,

the reduction in tiller survival, seed production and seed size, cause grain yield losses that

range from 13% to 45% on susceptible cultivars under severe epidemics (Everts and

Leath 1992; Lipps and Madden 1989). Powdery mildew infections can cause a reduction

6

in flour yield of 0.6% which represents roughly a $300,000 loss to a mill producing 1

million pounds of flour per day (Everts et al. 2001). The protein level in the grain can

also be affected by powdery mildew infections, where the protein content will increase

with shriveling of the grains (Johnson et al. 1979).

Powdery mildew epidemics are affected by temperature, moisture, and nutrient

availability. Cultural control methods that reduce the canopy moisture, such as lower

planting density, wider row spacing, and reduced irrigation may slow mildew

development, but will also likely reduce the yield potential of the wheat crop (Sharma et

al. 2004). Early nitrogen applications enhance the severity of powdery mildew epidemics

(Olesen et al. 2003). Most agricultural practices that reduce the primary inoculum, such

as plowing down crop debris and avoiding green bridges between winter and spring

forms of the same crop, are important in the delay of epidemic development. Fungicide

applications have been used to delay epidemics; however, chemical control methods

reqiure timely application, and can be erratic in effectiveness and costly (Bowen et al.

1991; Hardwick et al. 1994; Leath and Bowen 1989). Most chemical and cultural control

methods are used to delay inoculum spread or protect the crop until warmer temperatures

arrive (>25oC).

B. graminis has a sexual stage in which new genetic combinations of factors for

pathogenicity are produced. B. graminis is a heterothallic fungus and it has been

hypothesized that the genes for compatibility and pathogenicity are independent, which

facilitates the production of new pathotypes (Bruehl 1967). Annual sexual reproduction

7

and independent inheritance of virulence genes provides a constant supply of virulence

gene combinations in fit backgrounds. This has been evident from virulence surveys

conducted in the eastern United States (Niewoehner and Leath 1998; Leath and Heun

1990). The most recent survey found an increase in virulence to the widely deployed

resistance gene Pm17. Parks et al. (2008) reported the levels of intermediate virulence to

Pm17 increased from 9% to 20% during 2003 to 2005. This example illustrates the

importance of continued identification of new sources of resistance to provide effective

genetic control of powdery mildew.

Powdery mildew resistance in wheat

Breeding resistant cultivars is the most economical, environmentally safe, and

consistent method to reduce crop losses caused by powdery mildew in wheat. For these

reasons, host genetic resistance has been the most sought-after control method for

powdery mildew. With respects to powdery mildew, two types of genetic resistance are

available to plant breeders: race-specific or vertical resistance and race non-specific or

horizontal resistance (Bushnell 2002). Race-specific, qualitative, or monogenic

resistance is controlled by single major genes that are effective against certain powdery

mildew isolates with corresponding avirulence genes but are ineffective against other

isolates without the avirulence genes (Bennett 1984; Hsam and Zeller 2002).

Quantitative resistance is non-race-specific, is usually inherited polygenically, and is

8

usally associated with slow mildewing or reduced rates of infection (Hsam and Zeller

2004). Both types of resistance have been identified in wild and cultivated wheat

relatives.

Sources and breeding for powdery mildew resistance

Fifty three powdery mildew genes at 37 loci have been given formal gene

designations (Table 1) (Huang and Röder 2004; McIntosh et al. 2005; 2006, 2007,

personal communication). Another nine genes have been given temporary designations

and need further allelism tests to determine their relationship with known Pm genes

(Table 2). Researchers in many different countries have conducted surveys of collections

of T. aestivum and its wild relatives to identify readily useable sources of powdery

mildew resistance for breeding purposes (Leath and Heun 1990; Cox et al. 1992; Huang

et al. 1997b; Xie and Nevo 2008; Lebedeva and Peusha 2006). Only 24 of the named

resistance genes likely originated in the hexaploid T. aestivum germplasm pool. Another

22 resistance genes have been incorporated from other primary and secondary germplasm

pools, such as Ae. tauschii (Coss.) (DD; 2n=14), T. monoccocum L. (AA; 2n=14), T.

turgidum L. (AABB; 2n=28), and T. timopheevii (Zhuk.) Zhuk. (AAGG; 2n=28) (Chen

and Chelkowski 1999). Gene transfer from the primary and secondary gene pools is

accomplished with general breeding protocols such as direct hybridization, homologous

recombination, backcrossing and phenotypic selection (Hsam and Zeller 2002; Mujeeb-

9

Kazi and Rajaram 2002). More distantly related species in the tertiary gene pool of

wheat have been identified as the donors of seven Pm genes. Tertiary sources of

resistance genes include Ae. longissima (Schweinf. & Muschl. in Muschl.) Eig (SI),

Secale cereale L. and Haynaldia villosa (L.) Schur. The use of tertiary species requires

more advanced techniques that induce chromosome breakage and translocations,

including crossing with lines that possess mutated Ph1 genes, use gametocidal genes, and

ionizing radiation (Ceoloni et al. 1992; Hsam and Zeller 2002; Nasuda et al. 1998).

These techniques are coupled with embryo rescue to transfer resistance genes into

hexaploid wheat (Mujeed-Kazi and Rajaram 2002). Breeders should use caution when

using tertiary relatives as sources of powdery mildew resistance because transfers are not

always successful. The resistance gene Pm7, transferred from rye, has never been

commercially used since the wheat-rye translocation resulted in a dramatic yield

depression (Bennett 1984).

Race-specific resistance

Race-specific resistance utilizing major resistance genes which confer the

hypersensitive cell death response is the most common breeding strategy. This form of

resistance follows the gene-for-gene theory developed by Flor (1955). The gene-for-gene

theory states that for every resistance gene (R-gene) in the host plant there is a

corresponding avirulence gene (Avr-gene) in the pathogen. The relationship between the

10

host and pathogen determines whether there will be a compatible (susceptible) or

incompatible (resistant) reaction in the host. Generally, the R-genes in the host provide

immunity from the invading pest. However, race-specific resistance is ephemeral due to

changes in the pathogen population (Bennett 1984; Chen et al. 2005; Niewoehner and

Leath 1998; Robets and Caldwell 1970; Wang et al. 2005). The increase in virulence and

changes in virulence frequency are highly influenced by the resistance genes carried by

cultivars grown in a particular area. The selection pressures exerted by cultivars that

carry major resistance genes result in the rapid build-up of new powdery mildew

pathotypes with more effective virulence gene combinations. This contributes to a

decline in effectiveness of widely deployed race-specific resistance genes (Liu et al.

2001; Wang et al. 2005). Virulence surveys of the eastern United States have shown that

powdery mildew populations are able to carry a large number of virulence genes without

serious impact to their general fitness (Leath and Heun 1990; Niewoehner and Leath

1998; Parks et al. 2008). Because of the continual shifts towards higher virulence

frequencies and an increased number of virulence gene combinations in a fit background,

powdery mildew populations have overcome widely deployed resistance genes in a short

period of time (Parks et al. 2008).

11

Adult Plant Resistance

Quantitative resistance, also known as slow mildewing, partial resistance, non-

race-specific resistance, or adult plant resistance (APR), is expressed in adult plants but

not in seedlings. It is generally controlled by more than one gene. APR mechanisms

retard mycelial growth and the reproductive structures of the pathogen (Jakobson et al.

2006). Factors that contribute to powdery mildew control by APR include an increase in

the latent period (the time from infection until new spores are produced), reduced pustule

formation, and reduced production of spores, all of which reduce colony formation in the

absence of the hypersensitive response (Bushnell 2002). These factors decrease the

infection frequency and the number of secondary spores that successfully infect

neighboring plants (Shaner 1973). However, the current phenotypic selection methods

for APR are a time consuming process involving extensive and precise quantitative

measures (Gustafson and Shaner 1982; Mingeot et al. 2002). Several studies have been

conducted to identify quantitative trait loci (QTL) that influence APR in wheat. Keller et

al. (1999) identified 18 QTL that explained 77.2% of the phenotypic variance. Chantret

et al. (2000) found one race-specific gene MlRE, and another factor that accounted for

16-25% of the variation located on chromosome 5D. Liu et al. (2001) found three QTL

that explained 44% of the phenotypic variation for APR. Lillemo et al. (2006) evaluated

the partial resistance of powdery mildew in the CIMMYT line Saar and concluded that

the resistance was controlled by at least three genes. A common theme to most of the

12

above mentioned studies was that one or two major effect QTL were identified in

chromosomal regions of known Pm genes. However, all the researchers concluded that

the QTL were different from known Pm genes either by location of the QTL or by the

line of descent by which the resistance QTL were derived. Furthermore, it is not easy to

select plants with both APR and race-specific resistance because race-specific resistance

can confound QTL results (Mingeot et al. 2002). In contrast, the APR gene Pm38 has

been reported to confer a high level of broad-spectrum resistance, has been identified in

several genetic backgrounds, and is inherited as a gene complex which also confers

resistance to leaf rust, stem rust and leaf tip necrosis (Liang et al. 2006; Spielmeyer et al.

2005, 2008; Schnurbusch et al. 2004). Similar observations have been report for the

resistance gene Pm39, but this gene has only been identified in the cultivar Saar (Lillemo

et al. 2008).

Gene Postulation

The genetic basis of qualitative resistance has been determined by traditional

genetic analysis and the application of the gene-for-gene theory. Gene postulation

studies use techniques such as inheritance studies, molecular marker linkage analysis, and

allelism testing to determine the relationships between known and unknown Pm

resistance genes. Gene postulation employs a selected set of differential powdery mildew

isolates, each with different virulence/avirulence spectra, inoculated on detached leaf

13

segments of inbred lines with known Pm genes and lines with unknown resistance genes

(Hsam and Zeller 2002). By comparing the susceptible/resistant reaction patterns of the

known lines, researchers can postulate if the unknown line possesses a new Pm gene

(Chen and Chelkowski 1999; Hsam and Zeller 2002; Leath and Heun 1990). The

detached leaf test is an effective and quick method to identify major race-specific Pm

genes in wheat seedlings, but when the genes are not clearly expressed in the seedlings or

show quantitative resistance, the test must be carried out on adult plants. This has been

the traditional method to identify Pm genes. However, with the abundance of Pm genes,

the presence of more than one Pm gene in one line (resistance gene pyramid), and lack of

differential isolates to distinguish new unknown Pm genes, more advanced genetic

analysis is needed to characterize new unknown Pm genes.

Genetic localization

Genetic localization techniques, such as cytological and monosomic analysis,

have been used to identify where new Pm genes were introgressed into the wheat

genome. For introgression of resistance genes from tertiary relatives such as T. ovatum,

H. villosa, S. cereae, and Ae. longissima, cytogenetic techniques like C-banding have

been used. By observing the difference in C-banding patterns between the donor species

and wheat, researchers are able to identify the genetic region in which alien-introgessions

have occurred (Friebe and Heun 1989; Chen et al. 1995; Friebe et al. 1994; Hsam et al.

14

1995; Ceoloni et al. 1992; Heun and Friebe 1990). The largest disadvantage to

cytogenetic analysis is that the methods are highly technical, expensive, and time

consuming (Chen and Chelkowski 1999).

Monosomic analysis has been a powerful tool for Pm gene localization, especially

when the donor species is in the primary or secondary gene pool and hybridization can be

achieved. This technique employs the use of Chinese Spring (CS) monosomic lines

developed by Sears (1954). Monosomic analysis involves crossing the resistant line

under study with each of the CS monosomic lines (mono 1A-7D). The F2 progeny from

each cross are observed for their reaction to a powdery mildew isolate that is avirulent to

the donor line, but virulent on the CS monosomic lines. Crosses that significantly deviate

from the expected 3:1 (resistant:susceptible) ratio are identified as carrying the resistance

gene (See Singh 1967 for a more descriptive review of this process). Many Pm genes

have been identified though monosomic analysis including: Pm1 (Hsam et al. 1998),

Pm22 (Pm1e) (Peusha et al. 1996), Pm3 (Zeller et al. 1993), Pm5 (Hsam et al. 2001),

Pm13 (Ceoloni et al. 1992). Pm16 (Reader and Miller 1991), Pm2 and Pm19 (Lutz et al.

1995), Pm24 (Huang et al. 1997a), Pm28 (Peusha et al. 2000), and Pm32 (Hsam et al.

2003). Although monosomic analysis has been a popular technique used for genomic

localization of Pm genes, it does have some drawbacks. It is laborious in the amount of

crossing and the resolution at the chromosome level is low. Most monosomic analyses

only identify the chromosome which the gene is located and not the arm or

subchromosomal region in which the gene is located. In addition, when two or more

15

genes are linked on the same chromosome the power of this test is limited. Molecular

genetic mapping techniques were developed to alleviate these problems.

Genetic and molecular mapping

Molecular markers such as restriction fragment length polymorphisms (RFLPs),

random amplified polymorphic DNAs (RAPDs), amplified fragment length

polymophisms (AFLPs), microsatellites or simple sequence repeats (SSRs), sequence-

tagged sites (STS) and diversity array technology (DArT) have been used to genetically

and physically locate Pm genes in the wheat genome. Mapping is generally conducted in

F2 and backcross populations, near-isogenic lines (NILs), double haploids (DH), and

recombinant inbred lines (RILs) (Huang and Röder, 2004, Akbari et al. 2006). Some

genetic mapping techniques can be more difficult and less accurate than others, because

of the large genome size of wheat (16 x 109 bp), its polyploid nature, and sequence

conservation within genomes. The presence of the three related genomes (A, B, and D)

can increase the complexity of marker analysis, particularly with RFLPs, since three sets

of bands, one from each genome, will usually be obtained. This makes it difficult to

decipher which genome is responsible for the resistance gene (Landrige et al. 2001). This

problem can be alleviated though the use of CS aneuploid lines developed by Endo and

Gill (1996). Much of the early work with RFLP markers formed the framework for

linkage mapping with more specific molecular markers in wheat. Molecular markers can

16

alleviate false positive detection by monosomic analysis, such as in the case of the

misplacement of Pm1e (Pm22) (Singrün et al. 2003) and Pm16 (Chen et al. 2005) genes,

as well as locate genes to the subchromosomal level. Today most newly identified

powdery mildew genes/alleles have been mapped using polymerase chain reaction (PCR)

based molecular marker systems in coupling with CS aneuploid stocks (Huang and Röder

2004; Miranda et al. 2006; 2007; Perugini et al. 2008).

RFLP analysis

The first linkage maps of all seven homoeologous chromosomes were created

using RFLP markers (Langridge 2001; Röder et al. 1998). Most of the early maps were

created from populations derived from wide crosses and had limited application to

agronomic traits. Despite low polymorphism, many researchers in the 1990’s and early

2000’s used RFLPs to identify the chromosomal position of the known Pm genes.

Resistance genes Pm1 (Hartl et al. 1995), Pm4a (Ma et al. 1994), Pm6 (Tao et al. 2000),

Pm13 (Cenci et al. 1999), Pm26 (Rong et al. 2000), and Pm29 (Zeller et al. 2002) have

all been mapped using RFLP markers. However, the disadvantages of RFLPs have

limited their application to pragmatic breeding programs and their use has all but been

eliminated in wheat breeding because of their laborious nature and inconsistent gemone

location. These disadvantages outweight the advantages and the problems associated

17

with RFLPs were addressed with PCR based molecular marker systems (Langride et al.

2001).

RAPD analysis

Although RAPD markers are cheap and technically simple, their use has been

limited because of their dominant nature and unreliability. RAPD markers have been

used to map resistance genes Pm1 (Hu et al. 1997), Pm8 and Pm17 (Iqbal and Rayburn

1995), Pm21 (Qi et al. 1996), and Pm25 (Shi et al. 1998). RAPD technology has the

shortcoming of relatively low reproducibility between laboratories and even between

different PCR machines within the same laboratory. Because of their low level of

polymorphism and unreliability, RAPD markers have not been used for whole genome

linkage mapping in wheat and were only applied for linkage mapping to subchromosomal

regions where major resistance genes have been identified (Huang and Röder, 2004).

AFLP Analysis

The AFLP procedure is a sequence-arbitrary method based on PCR amplification

of restriction fragments generated by specific restriction enzyme combinations (Vos et al.

1995). AFLP analysis is a reliable and robust system to rapidly generate great numbers of

markers for the construction of high-density genetic maps, when no prior sequence

18

knowledge is available. A few powdery mildew resistance genes have been mapped with

the AFLP system which include Pm1c and Pm4a (Hartl et al. 1999), Pm17 (Hsam et al.

2000), Pm24 (Huang et al. 2000b), and Pm29 (Zeller et al. 2002). However, AFLP

markers tend to cluster around centromeric regions, are dominant in action, and are not

readily transferable between populations or laboratories. Many times SSR markers are

used to anchor AFLP linkage groups to their respective chromosomes.

SSR Analysis

Currently, genomic SSR markers are the primary and most popular system used

for linkage mapping and gene localization in wheat. SSRs are comprised of short repeat

units of 1-6 nucleotides. They are abundant, evenly dispersed throughout the genome,

and have higher levels of polymorphism than most other genetic markers in wheat.

These features, coupled with their ease of use, transferability between researchers, and

co-dominant nature, have made them a useful marker system (Langridge et al. 2001).

Röder et al. (1998) identified and mapped 279 SSR markers amplified by 230 primer sets.

They found SSRs fairly evenly distributed throughout the genome of common wheat. Of

the 279 loci identified, 93 mapped to the A genome, 115 to the B genome, and 71 to the

D genome. Currently, over 1000 SSRs mapped in many different reference populations

are available for wheat (Gupta et al. 2000; Guyomarc’h et al. 2002; Huang et al. 2003a;

Song et al. 2002; Stephenson et al. 1998). All of the most recently reported powdery

19

mildew resistance genes have been mapped with SSR markers: Pm30 (Liu et al. 2002),

Pm31 (Xie et al. 2003), Pm32 (Hsam et al., 2003), Pm33 (Zhu et al. 2005), Pm34

(Miranda et al., 2006), Pm35 (Miranda et al. 2007), Pm36 (Blanco et al. 2008) and Pm37

(Perugini et al. 2008).

Other marker systems

Since cloning of DNA or marker sequences is relatively inexpensive, RFLP,

RAPD and other markers are being converted to sequence tagged site (STS) markers.

Conversion of inconsistent markers to STS markers offers several advantages such as

higher and more consistent throughput, sharing of primer sequences, and the need for less

genomic DNA (Langridge et al. 2001). Development of STS markers from RAPD, RFLP

and cloned sequences has led to some successful markers being incorporated into marker

assisted selection programs (Cenci et al. 1999; Ji et al. 2008a; Ma et al. 2004; Mohler et

al. 2001; Neu et al. 2002; Tommasini et al. 2006; Yi et al. 2008).

DArT is a hybridization-based alternative to PCR, which can be performed on a

platform similar to microarrays (Akbari et al. 2006). Roughly 1,500 clones can be placed

on an array with this technology and individuals can be completely genotyped with one

array. It was reported that the polymorphism rate, distribution and uniqueness of the

DArT markers is similar to SSRs (Akbari et al. 2006). The DArT platform can be

adapted for bulked segregant analysis (BSA). DArT technology is able to detect a

20

polymorphic threshold for markers linked to the phenotypic character under study by

genotyping the bulk DNA pools (Wenzl et al. 2007). The current disadvantage of DArT

technology is the lack of sequence information on the clones. Because SSR or STS

markers cannot be developed from polymorphic markers, only the labs with the DArT

technology are able to use the identified markers. In addition, there has only been an

integrated DArT-SSR linkage map published thus far for tetraploid durum wheat

(Mantovani et al. 2008). However, with BSA, DArT analysis one is able to at least

identify the chromosome on which the locus governing phenotypic expression resides

(See Chapter 2).

Marker Assisted Selection (MAS)

Molecular markers that are closely linked to and segregate with the target genes

provide a useful tool for breeding programs. These markers allow the

detection/identification of the resistance gene(s) of interest without the need to perform

laborious phenotypic tests. This shortens the breeding time compared to traditional

breeding methods, and reduces the population size needed for recovery of a target

genotype (Bonnett et al 2005; Huang and Röder 2004). The essential requirements for a

marker(s) to be implemented in a MAS plant breeding program are: (1) they should be

economical to use and user-friendly, (2) they should co-segregate or be tightly linked to

the desired trait, (3) they should provide an efficient system for screening large

21

populations, (4) they should be highly reproducible across laboratories and exhibit

transferability among researchers, and (5) they should be co-dominant to differentiate

between homozygous and heterozygous individuals in a segregating population (Gupta et

al. 1999; Huang and Röder 2004; Langridge et al. 2001).

MAS has been useful in the development of Pm gene pyramids (Hsam and Zeller

2002; Huang and Röder 2004; Langridge et al. 2001). Liu et al. (2000) implemented

MAS to pyramid three two-gene combinations of powdery mildew resistance genes in a

Chinese wheat genotype. They used NILs with different resistance genes (Pm2, Pm4a,

and Pm21) to create segregating F2 populations. With the use of RFLP markers tightly

linked to the genes, they were able to identify three plants homozygous for both the Pm2

and Pm4a genes, and three plants homozygous for the Pm2 and Pm21 genes.

Combinations of several effective Pm resistance genes or even different alleles of the

same gene in a single cultivar could provide a more durable resistance package compared

to a single gene developed alone (Hsam and Zeller 2002; Huang and Röder 2004). The

best strategy to improve resistance to powdery mildew would be to combine resistance

genes based on APR mechanisms with major Pm genes that control recognition of the

pathogen (Keller et al. 1999).

22

Powdery Mildew Resistance Breeding at North Carolina State University

The North Carolina Small Grains breeding program initiated the development of

powdery mildew resistant germplasm in the late 1980’s. Thirteen germplasm lines have

been developed and released to broaden the genetic base of powdery mildew resistance

(Table 3). Diploid and tetraploid relatives of hexaploid wheat served as the donors of the

resistance genes. Embryo rescue techniques and subsequent backcrossing to the recurrent

parent cultivar Saluda (PI 480474) allowed for direct transfer of resistance genes in the

cases where diploid relatives were used as donors (Murphy et al. 1998, 1999a, 1999b).

Typical hybridization techniques and subsequent backcrossing to the recurrent parent

Saluda allowed for direct transfer of resistance genes where tetraploid emmer wheats

served as resistance gene donors (Murphy et al. 2002; Navarro et al. 2000). All thirteen

germplasm releases have shown unique susceptible/resistant reactions to powdery

mildew isolates identified for their respective virulence pattern and aggressiveness

(Murphy et al. 1998, 1999a, 1999b, 2002, 2004, Navarro et al. 2000).

With the use of segregating populations, molecular markers, BSA, differential

isolate tests, and nullitetrasomic and ditelosomic stocks, four out of the 13 germplasm

lines have been further studied to identify the gene action and precise chromosomal

location of the resistance gene. Shi et al. (1998) concluded that NC96BGTA5 carried a

single dominant gene (Pm25) donated from T. monococcum subsp. aegilopoides located

on chromosome 1A, linked to Pm3a. Srnić et al. (2005) characterized the resistance

23

genes possessed by NC96BGTA4 and NC99BGTAG11. They concluded that the genes

were both located on chromosome 7AL and developed AFLP and SSR markers for the

genes. More recently, Perugini et al. (2008) conducted an allelism test between the

resistance in NC99BGTAG11 and Pm1. They found that the resistance gene present in

NC99BGTAG11 was linked proximal to the Pm1 locus, and thus designated it Pm37.

SSR markers linked to resistance have been identified for NC96BGTA4 and

NC96BGTA6 on chromosome 7AL, but no allelism tests have been conducted to

determine their relationships (Miranda et al. 2007). Miranda et al. (2006) characterized

the resistance present in NC97BGTD7 and found that a single dominant gene was located

on the short arm of chromosome 5D. The gene was different from any named Pm gene

on chromosome 5D and thus designated Pm34. Similarly, Miranda et al. (2007)

characterized the resistance present in NC96BGTD3. This gene was also located on

chromosome 5D, but it was independent of Pm34, and thus it was designated Pm35. In

all these studies molecular markers were identified that were tightly linked to the genes

and should be useful in a MAS program.

Efforts are currently underway to pyramid the powdery mildew resistance genes.

Since all the germplasm lines had the same recurrent parent during their development,

limited inbreeding is needed to generate an agronomically uniform line. Currently, 12

lines have been developed with MAS, where lines homozygous for the markers linked to

the resistance gene have been selected and inbred. The lines have been characterized in

the field for one year and will undergo further agronomic evaluations in the upcoming

24

growing seasons. These germplasm lines are a valuable resource for the development of

genetic resistance in the southeastern United States since they are incorporated into an

adapted genetic background.

25

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McIntosh RA, Hart GE, Gale MD, Rogers WL (1998) Catalogue of gene symbols for wheat. In: Slinkard (eds), Proceedings of the 9th International Wheat Genet Symposium, University Extension Press, University of Saskatchewan, Saskatoon Canada pp 1-235 McIntosh RA, Hart GE, Devos KM, Gale MD, Rogers WJ, Dubcovsky J, Morris (2005) Catalogue of gene symbols for wheat: Annual supplement. http://wheat.pw.usda.gov/ggpages/wgc/2005upd.html. Cited 08 September 2008 McIntosh RA, Hart GE, Devos KM, Gale MD, Rogers WJ, Dubcovsky J, Morris (2006) Catalogue of gene symbols for wheat: Annual supplement. http://wheat.pw.usda.gov/ggpages/wgc/2006upd.html. Cited 08 September 2008 McIntosh RA, Hart GE, Devos KM, Gale MD, Rogers WJ, Dubcovsky J, Morris (2007) Catalogue of gene symbols for wheat: Annual supplement. http://wheat.pw.usda.gov/ggpages/wgc/2007upd.html. Cited 08 September 2008 Mingeot D, Chantret N, Baret PV, Dekeyser A, Boukhatem N, Sourdille P, Doussinault G, Jacquemin JM (2002) Mapping QTL involved in adult plant resistance to powdery mildew in the winter wheat line RE714 in two susceptible genetic backgrounds. Plant Breed 121:133-140 Miranda LM, Murphy JP, Marshall D, Cowger C, Leath S (2007) Chromosomal location of Pm35, a novel Aegilops tauschii derived powdery mildew resistance gene introgressed into common wheat (Triticum aestivum L.). Theor Appl Genet 114:1451-1456 Miranda LM, Murphy JP, Marshall D, Leath S (2006) Pm34: a new powdery mildew resistance gene transferred from Aegilops tauschii Coss. to common wheat (Triticum aestivum L.). Theor Appl Genet 113:1497-1504 Miranda LM, Perugini L, Srnić G, Brown-Guedira G, Marshall D, Leath S, Murphy JP (2007) Genetic mapping of a Triticum monococcum-derived powdery mildew resistance gene in common wheat. Crop Sci 47:2323-2329 Mujeeb-Kazi A, Rajaram S (2002) Transferring alien genes from related species and genera for wheat improvement. In: Curtis BC, Rajaram S, Gomez-Macpherson H (eds) Bread wheat. Improvement and production. FAO, Rome pp 1-17 Murphy JP, Leath S, Huynh D, Navarro RA, Shi A (1998) Registration of NC96BGTD1, NC96BGTD2 and NC96BGTD3 wheat germplasm resistant to powdery mildew. Crop Sci 38:570-571

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Murphy JP, Leath S, Huynh D, Navarro RA, Shi A (1999a) Registration of NC96BGTA4, NC96BGTA5 and NC96BGTA6 wheat germplasm. Crop Sci 39:883-884 Murphy JP, Leath S, Huynh D, Navarro RA, Shi A (1999b) Registration of NC97BGTD7 and NC97BGTD8 wheat germplasms resistant to powdery mildew. Crop Sci 39:884-885 Murphy JP, Navarro RA, Leath S (2002) Registration of NC99BGTAG11 wheat germplasm resistant to powdery mildew. Crop Sci 42:1382 Mohler V, Hsam SLK, Zeller FJ, Wenzel G (2001) An STS marker distinguishing the rye-derived powdery mildew resistance alleles at the Pm8/Pm17 locus of common wheat. Plant Breed 120:448-450 Mohler V, Zeller FJ, Wenzel G, Hsam SLK (2005) Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.). 9. Gene MlZec1 from the Triticum dicoccoides-derived wheat line Zecoi-1. Euphytica 142:161-167 Mori N, Moriguchi T, Nakamura C (1997) RFLP analysis of nuclear DNA for study of phylogeny and domestication of tetraploid wheat. Genes Genet. Syst 72:153-161 Nasuda S, Friebe B, Gill BS (1998) Gametocidal genes induce chromosome breakage in the interphase prior to the first mitotic cell division of the male gametophyte in wheat. Genetics 149:1115-1124 Navarro RA, Murphy JP, Leath S, Shi A (2000) Registration of NC97BGTAB9 and NC97BGTAB10 wheat germplasm lines resistant to powdery mildew. Crop Sci 40:1508-1509 Nematollahi G, Mohler v, Wenzel G, Zeller FJ, Hsam SLK (2008) Microsatellite mapping of powdery mildew resistance allele Pm5d From common wheat line IGV1-455. Euphytica 159:307-313 Neu C, Stien N, Keller B (2002) Genetic mapping of the Lr20-Pm1 resistance locus reveals suppressed recombination on chromosome arm 7AL in hexaploid wheat. Genome 45:737-744 Niewoehner AS, Leath S (1998) Virulence of Blumeria graminis f. sp. tritici on winter wheat in the eastern United States. Plant Dis 82:64-68

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Song W, Xie H, Liu Q, Xie C, Ni Z, Yang T, Sun Q, Liu Z (2007) Molecular identification of Pm12-carrying introgression lines in wheat using genomic and EST-SSR markers. Euphytica 158:95-102 Song QJ, Fickus EW, Cregan PB (2002) Characterization of trinucleotide SSR motifs in wheat. Theor Appl Genet 104:286-293 Spielmeyer W, McIntosh RA, Kolmer J, Lagudah ES (2005) Powdery mildew resistance and Lr34/Yr18 genes for durable resistance to leaf and stripe rust cosegregate at a locus on the short arm of chromosome 7D of wheat. Theor Appl Genet 111:731-735 Spielmeyer W, Singh RP, McFadden H, Wellings CR, Huerta-Espino J, Kong X, Appls R, Lagudah ES (2008) Fine scale genetic and physical mapping using interstitial deletion mutants of Lr34/Yr18: a disease resistance locus effective against multiple pathogens in wheat. Theor Appl Genet 116:481-490 Srnić G, Murphy JP, Lyerly JH, Leath S, Marshall DS (2005) Inheritance and chromosomal assignment of powdery mildew resistance genes in two winter wheat germplasm lines. Crop Sci 45:1578-1586 Stephenson P, Bryan G, Kirby J, Collins A, Devos K, Busso C, Gale M (1998) Fifty new microsatellite loci for the wheat genetic map. Theor Appl Genet 97:946-949 Sun XL, Liu D, Zhang HQ, Huo NX, Zhou RH, Jia JZ (2006) Identification and mapping of two new genes conferring resistance to powdery mildew from Aegilops tauschii (Coss.) Schmal. J Integr Plant Biol 48:1204-1209 Tao W, Liu D, Liu J, Feng Y, Chen P (2000) Genetic mapping of the powdery mildew resistance gene Pm6 in wheat by RFLP analysis. Theor Appl Genet 100:564-568 Te Beest DE, Paveley ND, Shaw MW, van den Bosch F (2008) Disease-weather relationships for powdery mildew and yellow rust on wheat. Phytopathology 98:609-617 The TT, McIntosh RA, Bennett FGA (1979) Cytogenetical studies in wheat. IX. Monosomic analysis, telocentric mapping and linkage relationship of gene Sr1, Pm4, and Mle. Aust J Biol Sci 32:115-125 Tommasini L, Yahiaoui N, Srichumpa P, Keller B (2006) Development of functional markers specific for seven Pm3 resistance alleles and their validation in the bread wheat gene pool. Theor Appl Genet 114:165-175

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Tosa Y, Sakai K (1990) Analysis of the resistance of Aegilops squarrosa to the wheatgrass mildew fungus by using the gene-for-gene reationship. Theor Appl Genet 81:735-739 Tosa Y, Tsijimoto H, Ogura H (1987) A gene involved in the reistance of wheat to wheatgrass powdery mildew fungus. Genome 29:850-852 Vos P, Bleeker M, Reijans M, Vandelee T, Hornes M, Frijter A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP – a new technique for DNA-fingerprinting. Nucleic Acids Res 23:4407-4414 Wang ZL, Li LH, He ZH, Duan XY, Zhou YL, Chen XM, Lillemo M, Singh RP, Wang H, Xia XC (2005) Seedling and adult-plant resistance to powdery mildew in Chinese bread wheat cultivars and lines. Plant Dis 89:457-463 Wenzl P, Raman H, Wang J, Zhou M, Huttner E, Kilian A (2007) A DArT platform for quantitative bulked segregant analysis. BMC Genomics 8:196-205 Wiese, MV (1987) Compendium of wheat diseases second edition. American Phytopathological Society, St. Paul Minnesota pp 30 Wright DP, Baldwin BC, Shephard MC, Scholes JD (1995) Source-sink relationships in wheat leaves infected with powdery mildew. I. Alterations in carbohydrate metabolism. Physiological and Molecular Plant Pathology 47:237-253 Xie C, Sun Q, Ni Z, Yang T, Nevo E, Fahima T (2003) Chromosomal location of a Triticum dicoccoides-derived powdery mildew resistance gene in common wheat by using microsatellite markers. Theor Appl Genet 106:341-345 Xie C, Sun Q, Ni Z, Yang T, Nevo E, Fahima T (2004) Identification of resistance gene analogue markers closely linked to wheat powdery mildew resistance gene Pm31. Plant Breed 124:198-200 Xie W, Nevo E (2008) Wild emmer: genetic resources, gene mapping and potential for wheat improvenment. Euphytica (online) Yahiaoui N, Brunner S, Keller B (2006) Rapid generation of new powdery mildew resistance genes after wheat domestication. Plant J 47:85-98 Yao G, Zhang J, Yang L, Xu H, Jiang Y, Xiong L, Zhang C, Zhang Z, Ma Z, Sorrells M (2007) Genetic mapping of two powdery mildew resistance genes in einkorn (Triticum monococcum L.) accessions. Theor Appl Genet 114:351-358

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Yi YJ, Liu HY, Haung XQ, AN LZ, Wang F, Wang ZL (2008) Development of molecular markers linked to the wheat powdery mildew resistance gene Pm4b and marker validation for molecular breeding. Plant Breeeding 127:116-120 Zeller FJ, Kong L, Hartl L, Mohler V, Hsam SLK (2002) Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.) 7. Gene Pm29 in line Pova. Euphytica 123:187-194 Zeller FJ, Lutz J, Stephan U (1993) Chromosome location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L.) 1. Mlk and other alleles at the Pm3 locus. Euphytica 68:223-229 Zhu Z, Zhou R, Kong X, Dong Y, Jia J (2005) Microsatellite markers linked to 2 powdery mildew resistance genes introgressed from Triticum carthlicum accession PS5 into common wheat. Genome 48: 585-590 Zulu JN, Farrar JF, Whitbread R (1991) Effects of phosphate supply on the phosphorus status, dry mass, and photosynthesis of wheat infected with powdery mildew. New Phytol 118:453-461

39

Table 1.1: Formally designated powdery mildew resistance genes, their chromosomal location, source of the resistance, first reference for each gene, subsequent PCR based linked molecular markers type(s), and reference for marker(s) Locus Chromosome

Location Source Original

Reference Linked PCR based Marker Type

Marker Referencb

Pm1a 7AL T. aestivum Sears and Briggle 1969

SSR, STS Neu et al. 2002

Pm1b 7AL T. monococcum Hsam et al. 1998 Pm1c (Pm18) 7AL T. aestivum Hsam et al. 1998 Pm1d 7AL T. spelta Hsam et al. 1998 Pm1e (Pm22) 7AL T. aestivum Singrün et al. 2003 SSR OPm2 5DS Ae. tauschii McIntosh and

Baker 1970; Lutz et al. 1995

Pm3a 1AS T. aestivum Briggle and Sears 1966

STS Tommasini et al. 2006

Pm3b 1AS T. aestivum Briggle 1966 STS Tommasini et al. 2006

Pm3c 1AS T. aestivum Briggle 1966 STS Tommasini et al. 2006

Pm3d 1AS T. aestivum Zeller et al. 1993 STS Tommasini et al. 2006

Pm3e 1AS T. aestivum Zeller et al. 1993 STS Tommasini et al. 2006

Pm3f 1AS T. aestivum Zeller et al. 1993 STS Tommasini et al. 2006

Pm3g 1AS T. aestivum Yahiaoui et al. 2006

STS Tommasini et al. 2006

40

Pm4a 2AL T. dicoccum The et al. 1979 STS Ma et al. 2004 Pm4b 2AL T. carthlicum The et al. 1979 STS Yi et al. 2008 Pm4c (Formally Pm23)

2AL T. aestivum Hao et al. 2008 SSR O

Pm5a 7BL T. dicoccum Law and Wolf 1966

Pm5b 7BL T. aestivum Hsam et al. 2001 Pm5c 7BL T. sphaerococcum Hsam et al. 2001 Pm5d 7BL T. aestivum Hsam et al. 2001 SSR Nematollahi et al.

2008 Pm5e 7BL T. aestivum Huang et al. 2003b SSR O Mlxbd (Pm5 allele)

7BL T. aestivum Huang et al. 2000a

Pm6 2B T. timopheevii Jorgensen 1973 STS Ji et al. 2008a Pm7 TABS.2RL S. cereale Friebe et al. 1994 Pm8 T1BL.1RS S. cereale Hsam and Zeller

1997 STS Mohler et al. 2001

Pm9 7A T. aestivum Hsam et al. 1998 Pm10a 1D T. aestivum Tosa et al. 1987 Pm11a 6BS T. aestivum Tosa et al. 1987 Pm12 6BS Ae. speltoides Jia et al. 1996 SSR Song et al. 2007 Pm13 T3BL.3S Ae. longissima Ceoloni et al.1992 STS Cenci et al. 1999 Pm14a 6B T. aestivum Tosa and Sakai

1990

Pm15a 7DS T. aestivum Tosa and Sakai 1990

Pm16 5BS T. dicoccoides Reader and Miller 1991

SSR Chen et al. 2005

Pm17 T1AL.1RS S. cereal Heun et al. 1990 STS Mohler et al. 2001

Table 1.1 Continued

41

Pm19 7D Ae. tauschii Lutz et al. 1995 Pm20 T6BS.6RL S. cereal Friebe et al. 1994 Pm21 T6AL.6VS H. villosa Chen et al. 1995 SCAR Lui et al. 1999 Pm24 1D T. aestivum Huang et al. 1997a

Huang et al. 2000b SSR Huang et al. 2000b

Pm25 1A T. monococcum Shi et al. 1998 Pm26 2BS T. dicoccoides Rong et al. 2000 Pm27 6B T. timopheevii Järve et al. 2000 SSR O Pm28 1B T. aestivum Peusha et al. 2000 Pm29 7DL Ae. ovata Zeller et al. 2002 AFLP O Pm30 5BS T. dicoccoides Lui et al. 2002 SSR O Pm31 (mlG) 6AL T. dicoccoides Xie et al. 2003;

Xie et al. 2004 SSR/RGA O

Pm32 T1BL.1SS Ae. speltoides Hsam et al. 2003 Pm33 2BL T. cathlicum Zhu et al. 2005 SSR O Pm34 5DS Ae. taushii Miranda et al.

2006 SSR O

Pm35 5DS Ae. taushii Miranda et al. 2007

SSR O

Pm36 5BL T. dicoccoides Blanco et al. 2008 SSR-EST O Pm37 7AL T. timopheevii Perugini et al.

2007 SSR O

Pm38 7DS T. aestivum Spielmeyer et al. 2005

SSR O

Pm39 1BL T. aestivum Lillemo et al. 2008 SSR O a Resistant to Blumeria graminis f. sp. agropyri b O = Original Reference

Table 1.1 Continued

42

Table 1.2: Temporary designated wheat powdery mildew resistance genes, their chromosomal location, source, and reference Locus Chromosome

Location Source Reference

mlRD30 7AL T. aestivum Singrün et al. 2004 mlZec 2BL T. dicoccoides Mohler et al. 2005 mlRE 6AL T. dicoccum Chantret et al. 2000 PmU 7AL T. urartu Qiu et al. 2005 Mlm2033 7AL T. monococcum Yao et al. 2007 Mlm80 7AL T. monococcum Yao et al. 2007 mlWI72 7AL T. dicoccoides Ji et al. 2008b PmY201 5DL Ae. tauschii Sun et al. 2006 PmY212 5DL Ae. tauschii Sun et al. 2006

43

Table 1.3 NC State wheat powdery mildew resistant germplasm lines, pedigree, source of the resistance, chromosomal location of the resistance gene, and gene desigantation NC Germplasm Line Pedigree Source of Resistance Chromosomal

Location Gene Designation

NC96BGTD1 (NC-D1) Saluda*3/TA2570 Ae. tauschii 7DS

NC96BGTD2 (NC-D2) Saluda*3/TA2481 Ae. tauschii UNKNOWN

NC96BGTD3 (NC-D3) Saluda*3/TA2377 Ae. strangulata 5DL Pm35

NC96BGTA4 (NC-A4) Saluda*3/PI221414 T. monococcum 7AL

NC96BGTA5 (NC-A5) Saluda*3/PI427662 T. aegilopoides 1AS Pm25

NC96BGTA6 (NC-A6) Saluda*3/PI427772 T. aegilopoides 7AL

NC97BGTD7 (NC-D7) Saluda*3/TA2492 Ae. tauschii 5DL Pm34

NC97BGTD8 (NC-D8) Saluda*3/TA2466 Ae. tauschii PROBABLY 5DL

NC97BGTAB9 (NC-AB9) Saluda*2//Cando/PI471735 T. dicoccoides UNKNOWN

NC97BGTAB10 (NC-AB10) Saluda*3//Ward/PI471746 T. dicoccoides 2BL

NC99BGTAG11 (NC-AG11)

Saluda*3/PI427315

T. timopheevii

7AL

Pm37

44

NC06BGTAG12 (NC-AG12) Saluda*3/PI538457 T. timopheevii 7AL

NC06BGTAG13 (NC-AG13) Saluda*3/PI427442 T. timopheevii 7AL

Table 1.3 Continued

45

Chapter II

MlNCD1: a novel Aegilops tauschii derived powdery mildew resistance gene

identified in common wheat

46

Abstract

Powdery mildew is a major fungal disease in wheat especially in cool maritime

climates. A novel wheat powdery mildew resistance gene present in the germplasm line

NC96BGTD1 was genetically characterized as a monogenic trait in greenhouse and field

trials using F3 families from a NC96BGTD1 / ‘Saluda’ cross. SSR markers were used to

map and tag the resistance gene present in NC96BGTD1. Two dominant SSR markers

flanking the resistance gene were identified. Xgwm635 and Xcfd41 mapped 5.3 cM distal

and 20 cM proximal to the resistance gene, respectively. These SSR markers were

previously mapped to the short arm of chromosome 7D and their positions were

confirmed using Chinese Spring aneuploid and deletion stocks. Only the adult plant

resistance gene Pm38 has been mapped to the short arm of chromosome 7D, but at a

different locus. The resistance gene described herein should be temporarily designated

MlNCD1.

47

Introduction

Powdery mildew, caused by Blumeria graminis (syn. Erysiphe graminis) f. sp.

tritici, is an economically important disease of wheat (Triticum aestivum L.) in cool

maritime environments. Grain yield can be reduced by as much as 48% in severe

epidemics (Everts and Leath 1992) due to reduced tiller survival, kernels per head, and

kernel weight (Bowen et al. 1991). Powdery mildew can negatively affect end-use

quality parameters such as flour yield, protein content, and softness equivalent (Everts et

al. 2001). Powdery mildew infections cause a decrease in photosynthetic activity coupled

with a reduction in transpiration, which reduce the carbohydrates available for grain fill

(Shtienberg 1992).

Control of powdery mildew through cultural and chemical approaches can be

detrimental to yield, erratic in effectiveness, and costly (Hardwick et al. 1994; Leath and

Bowen 1989). Thus, genetic resistance is a desirable control strategy. The most

commonly applied form of genetic resistance is race-specific or qualitative resistance

(Huang and Roder 2004). Fifty-six powdery mildew genes at 40 loci have been formally

designated (Huang and Roder 2004; McIntosh et al. 2005, 2006, 2007, personal

communication). Frequent changes in virulence frequencies in the powdery mildew

population make it difficult to control the pathogen with any single resistance gene for a

long period of time (Leath and Heun 1990; Niewoehner and Leath 1997). A recent

virulence survey of the eastern United States found the number of virulence genes in the

powdery mildew population continued to increase, and there was a shift towards higher

48

virulence frequencies on the widely deployed Pm17 gene derived from Secale cereale

(Parks et al. 2008). Thus, it is necessary to identify new sources of mildew resistance for

incorporation into adapted backgrounds.

Many race-specific resistance genes were identified in the primary and secondary

wheat gene pools (Huang and Roder 2004). Direct gene transfer between the primary

and secondary gene pools is accomplished by direct hybridization, homologous

recombination, and embryo rescue techniques (Mujeeb-Kazi and Rajaram 2002).

Aegilops tauschii Coss. (2n = 2x = 14; DD) is a diploid wheat progenitor and donor of the

D genome to common wheat (2n = 6x = 42; AABBDD). Many accessions of Ae. tauschii

were reported to exhibit resistance to powdery mildew (Cox et al. 1992; Lutz et al. 1994).

Resistance genes Pm2, Pm19 (Lutz et al. 1995), Pm34 (Miranda et al. 2006) and Pm35

(Miranda et al. 2007) are derived from Ae. tauschii and introgressed into the D genome of

common wheat.

The characterization of major resistance genes in germplasm lines, coupled with

the identification of closely linked molecular markers, simplifies activities such as

cultivar development (Bonnett et al. 2005), near-isogenic line development (Zhou et al.

2005), and pyramiding resistance genes into one cultivar. Liu et al. (2000) provided a

protocol for pyramiding resistance genes into a single genotype and showed that wheat

lines with multiple resistance genes exhibited broad-spectrum resistance to powdery

mildew.

49

The germplasm line NC96BGTD1 was released by North Carolina State

University in 1996. It continues to exhibit a high level of resistance to naturally

occurring field isolates of powdery mildew in the eastern United States. The objectives

of this research were to genetically characterize the powdery mildew resistance in

NC96BGTD1, identify SSR markers flanking the resistance gene, and to determine if the

gene is a novel Ae. tauschii-derived powdery mildew resistance gene.

50

Materials and Methods

Plant Material

The powdery mildew resistant germplasm line NC96BGTD1 (PI 597348) was crossed

with the susceptible cultivar Saluda (PI 480474). NC96BGTD1, hereafter referred to as

NC-D1, is a BC2F6-derived line with the pedigree Saluda*3/TA 2570 (Murphy et al.

1998). TA 2570 is an Ae. tauschii subsp. tauschii accession collected in Armenia.

Saluda is a soft red winter wheat developed and released by Virginia Polytechnic Institute

and State University (Starling et al. 1986). Saluda possesses the powdery mildew

resistance gene Pm3a, which is not effective against naturally occurring powdery mildew

populations in North Carolina (Niewoehner and Leath 1998; Parks et al. 2008). The NC-

D1 / Saluda F1 hybrid and subsequent F2 seeds were used to produce F3 families for

greenhouse and field evaluations. A reserve F2 population was used to determine the

gene action of the powdery mildew resistance in NC-D1.

Greenhouse evaluations

One hundred F3 families were evaluated for reaction to powdery mildew during the

spring of 2002. An experimental unit was two 10 cm pots each planted with five seeds of

each line. A completely randomized design with one replication was used, where pot

51

pairs were randomly assigned a position in the greenhouse and two pots containing the

parental lines NC-D1 and Saluda were included at 10-pot intervals as controls. Each

control pot contained two plants. Seeds of the parental lines were derived from selfed

progenies of the original plants used to produce the population under study. The seeds

were planted in a mixture of Metro-Mix 200 (Scotts-Sierra Horticultural Products Co.,

Marysville, OH), soil, and sand in a 50:40:10 ratio, respectively, with the addition of

three grams Osmocote (14N:14P:14K) (The Scotts Company LLC) fertilizer. The

temperature was maintained at 24oC/20oC (day/night). Plants were grown under plentiful

natural light supplemented with artificial High Intensity Discharge 1000W lights.

The experiment was inoculated at Feekes growth stage 2 to 3 by gently shaking

conidia from leaves of infected Saluda plants onto leaves of the F3 families and control

plants. The inoculum was collected from Saluda plants grown at the Cunningham

Research and Education Center at Kinston, NC, in April 2001. The clearest differences

between resistant and susceptible reactions were evident 12 to 20 days post inoculation.

Disease evaluations were conducted when Saluda control plants showed intense signs and

symptoms of powdery mildew infection. The disease severity evaluation was on a scale

from 0 to 9 as described by Leath and Heun (1990), where: 0 = immune, no visible signs

of infection; 1-3 = resistant, increasing from 1) flecks, no necrosis, to 2) necrosis, to 3)

chlorosis, while the amount of mycelium went from none to a detectable amount; 4-6 =

intermediate, chlorotic areas decreasing in amount while mycelium and conidial

production increased from slight to moderate; 7-9 = susceptible, with increasing amount,

52

size and density of mycelium and conidia to a fully compatible reaction. The F3 families

with disease reactions similar to NC-D1 were classified as ‘resistant’ and those similar to

Saluda were classified as ‘susceptible’. Families that exhibited both resistant and

susceptible plants were classified as ‘segregating’. Chi-square tests were conducted to

test the goodness of fit between observed and expected segregation ratios (Snedecor and

Cochran 1956).

An additional 100 F2 seedlings from the cross NC-D1 / Saluda were grown in the

greenhouse in 6inch deep Ray Leach “Cone-tainers” (Stuewe and Sons, Inc., Tangent,

Oregon, USA) using the protocol described above. Plants were inoculated with the

powdery mildew isolate ‘Yuma’, which is virulent on Saluda (Pm3a) and avirulent on

NC-D1. Yuma is an isolate maintained by the USDA-ARS plant pathology lab at North

Carolina State University in Raleigh, NC. Ten seedlings of Saluda and NC-D1 were

included in evaluation as checks. Chi-square test was conducted to test the goodness of

fit between observed and expected segregation ratios (Snedecor and Cochran 1956).

Field evaluation

One hundred-ninety-three F3 families were planted at the Cunningham Research

and Education Center at Kinston, NC in October 2001. The 100 families tested in the

greenhouse were included in the field test. The experimental design was a randomized

complete block with two replications. An experimental unit consisted of a single row

53

sown with 30 to 60 seeds and the rows were spaced 31 cm apart. Each family was

randomly assigned a row in each block. NC-D1 and Saluda were included as controls

every 40 plots. Twelve differential genotypes with previously identified Pm genes were

included in each replication: Axminister (Pm1), Ulka (Pm2), Asosan (Pm3a), Chul

(Pm3b), Sonora (Pm3c), Michigan Amber (Pm3f), Yuma (Pm4), Hope (Pm5), Coker 747

(Pm6), Transec (Pm7), Federation*4/Karvkaz (Pm8), Amigo (Pm17), and the cultivar

Chancellor (no Pm genes). A 1.2-m border of Saluda surrounded the experiment.

Irrigation, fertilization, and other agronomic practices were conducted as needed

following standard management practices for North Carolina (Weisz 2000).

Disease reaction evaluations were initiated at the end of March 2002 when all

Saluda rows showed uniform powdery mildew infection. Plants were between Feekes

growth stage 9 and 10.1. Flag leaf minus 2 was evaluated using the modified scale of

Leath and Heun (1990) on 12 to 24 random plants per plot. All plots were evaluated

three times (March 29, April 12, and April 18), and disease reactions were the same for

each plat on the latter two dates. Chi-square tests were conducted to examine the

goodness of fit between observed and expected segregation ratios (Snedecor and Cochran

1956).

54

Detached leaf test

Powdery mildew disease reactions were evaluated in the laboratory using a

detached leaf technique. Primary leaf segments (1.5 cm) were floated on 0.5% water

agar amended with 50 mg l-1 benimidazole in plastic plates. Lines possessing Pm3a,

Pm19, NC-D1 and the susceptible control Chancellor were inoculated with 5 different

powdery mildew isolates collected from wheat fields in the eastern region of North

Carolina. Plates were placed in a growth chamber maintained at 18oC, 85% relative

humidity and with a photoperiod of 12 h. The disease reactions were based on the same

scale used for the greenhouse and field evaluations. Reactions were recorded 10 days

post inoculation.

Markers and linkage analysis

Genomic DNA was extracted from leaf tissue of 193 F2 plants that gave rise to the

F3 families by the CTAB procedure described by Stein et al. (2001). Wheat SSR primers

were synthesized according to the sequence published in the GrainGenes database

(http://wheat.pw.usda.gov), with all forward primers modified to include the M13

sequence (CACGACGTTGTAAAACGAC-) at the 5’ end for labeling purposes (Schulke

2000). PCR reaction conditions and visualization were conducted as described by

Miranda et al. (2006). DNA from 10 homozygous resistant and 10 homozygous

55

susceptible individuals was pooled to generate resistant and susceptible bulks for bulk

segregant analysis (BSA) (Michelmore et al. 1991). Markers found to be polymorphic

between NC-D1 and Saluda and the resistant and susceptible bulks were tested for F2

segregation and linkage with the resistant phenotype. Once linkage was detected,

additional markers in the chromosomal region were evaluated for development of the

linkage map.

Chi-squared analyses were used to test for expected 3:1 segregation for dominant

markers and 1:2:1 segregation for codominant markers. Linkage analysis was conducted

using MAPMAKER/EXP (version 3.0b) (Lincoln et al. 1993). Default mapping

functions were used for analysis. Maximum likelihood estimates confirmed map orders

of the wheat consensus map (Somers et al. 2004).

Chromosome locations of linked markers were confirmed using ‘Chinese Spring’

(CS) and chromosomal 7D-related CS aneuploids kindly provided by the Wheat Genetic

Resource Center at Kansas State University. These aneuploid lines were: nullisomic 7D-

tetrasomic 7B (N7D-T7B), nullisomic 7B-tetrasomic 7D (N7B-T7D), ditelosomic 7DS

(Dt7DS), ditelosomic 7DL (Dt7DL). In addition, three CS deletion stocks with deletions

in the short arm of chromosome 7D were utilized: 7DS-5 (FL = 0.36), 7DS-1 (FL= 0.37),

7DS-4 (FL = 0.61) and 7DL-6 (FL = 0.10 of the long arm). The designation “FL”

indicates the fraction length of the chromosome arm that was present, i.e., the line 7DS-5

has retained the proximal 36% of the short arm of chromosome 7D (Endo and Gill 1996;

Sourdille et al. 2004).

56

Results

Greenhouse and field evaluations

NC-D1 and Saluda had mean disease severity scores of 4.6±0.47 and 7.0±0.00,

respectively, in the greenhouse evaluation. The NC-D1 disease severity score for the

greenhouse test was in the intermediate range and the isolate utilized was more

aggressive in the greenhouse than the field. Nevertheless, there was uniformity within

the resistant families with all plants scoring 4 to 5 and all plants scoring 7 within the

susceptible families. The families scored as segregating had a mixture of plants scored

from 4 to 7. The observed seedling segregation ratio in the greenhouse test for the NC-

D1 / Saluda F3 population fitted the expected ratio for monogenic inheritance of the

powdery mildew resistance present in NC-D1 (Table 1).

NC-D1 and Saluda had average disease severity scores of 1.4±0.53 and 6.6±0.52,

respectively, in the field evaluation. The observed segregation in the field also fit the

expected segregation for monogenic inheritance of the powdery mildew resistance

present in NC-D1 (Table 1). Of the 100 families evaluated in the greenhouse and field,

seven were classified as segregating in the field and susceptible in the greenhouse, two

were classified as segregating in the field and resistant in the greenhouse, seven were

classified as resistant in the field and segregating in the greenhouse, and two were

classified as susceptible in the field and segregating in the greenhouse. Differences in

57

agreement between the field and greenhouse evaluations were likely due to sample size

and the difference in environmental conditions as mentioned earlier. Because of the large

sample size, the field rating were utilized when ther were discrepancies between

greenhouse and field results. The mean field disease severity score for NC-D1 was

significantly less than all 12 differential lines that possessed a major Pm gene, except for

Pm17 (data not shown) indicating that NC-D1 processes a powdery mildew resistance

gene effective against natural infection by powdery mildew isolates of the southeastern

US.

Marker Analysis

Wheat SSR markers were used to identify the chromosomal region in which the

powdery mildew resistance gene was located in NC-D1. Fifty eight of 193 D-genome

specific SSR markers evaluated for polymorphism between NC-D1 and Saluda were

polymorphic. These markers were tested in a bulk segregant analysis (BSA) using

pooled DNA from the F2 plants that gave rise to 10 homozygous resistant and 10

homozygous susceptible F3 families. Six markers reported to be located on the short arm

of chromosome 7D (Somers et al. 2004) were polymorphic between the resistant and

susceptible bulks. Linkage analysis located markers Xgwm635 and Xcfd41 5.3cM distal

and 20cM proximal, respectively, to the powdery mildew resistance gene present in NC-

D1 (Figure 1). Both flanking markers are dominant for the Saluda allele. Xgwm635 and

58

Xcfd41 produced bands of 117 bp and 181 bp respectively, and are linked in repulsion

with the NC-D1 allele. Additional chromosome 7D markers, Xwmc506, Xgdm88,

Xgdm145 and Xcfd66, were used to confirm the order of the markers and location of the

powdery mildew resistance gene in NC-D1 (Figure 1). Xwmc506, Xgdm145, Xcfd41, and

Xgwm635 were dominant for the Saluda allele, were null for the NC-D1 allele, and

segregated in the expected 1:3 ratio. The markers Xgdm88 and Xcfd66 were co-dominant

and segregated in the expected 1:2:1 ratio (Table 2).

The marker order and distance between markers was in close agreement with the

Wheat Consensus Map (Somers et al. 2004). CS aneuploid and deletion stocks were used

to confirm the chromosomal locations of the SSR markers on 7DS. Marker Xgwm635

amplified a band of 117 bp in Chinese Spring and the ditelosomic 7DS line (Dt7DS), but

no products were observed in the ditelosomic 7DL line (Dt7DL) or nullisomic 7D-

tetrasomic 7B (N7D-T7B) line (Figure 2). Three CS deletion stocks were used to

determine the deletion interval in which the resistance gene was located. SSR marker

Xgwm635 did not produce the expected fragment in any line indicating that the marker,

and likely also the resistance gene possessed by NC-D1, were located in the terminal

39% of the chromosome arm, distal to the breakpoint of deletion line 7DS-4 ( FL = 0.61)

(Figure 2). Similar results were observed for Xwmc506, which did not produce a band in

any of the deletion lines tested, as would be expected since it mapped distal to Xgwm635.

Markers Xcfd41 and Xcfd66 produced bands of expected size in CS and 7DS-5, indicating

59

these markers were located just distal to the centromere in the deletion bin C-7DS5-0.36,

which is in agreement with Sourdille et al. (2004).

60

Discussion

Both the greenhouse and field evaluations provided strong evidence that the

powdery mildew resistance in NC-D1 is controlled by a single dominant gene, which is

typical of the race-specific powdery mildew genes identified thus far (Huang and Roder

2004). NC-D1 has exhibited high levels of resistance in field evaluations in North

Carolina through 2008 and would be an effective parent if used in breeding programs.

Four Pm genes have been identified on wheat chromosome 7D. Tosa and Sakai

(1990) identified the Ae. tauschii-derived powdery mildew resistance gene Pm15 on

chromosome 7DS using monosomic analysis. The Pm15 gene is effective only against

Blumeria graminis f. sp. agropyri (and its hybrids), the causal organism of wheatgrass

powdery mildew, and is not known to be effective against wheat powdery mildew. Lutz

et al. (1995) assigned Pm19 to chromosome 7D using monosomic analysis. This gene

was identified in the synthetic wheat line ‘XX186’, but no chromosomal arm designation

was given. A detached leaf test was conducted using five different powdery mildew

isolates between NC-D1 and XX186. The line XX186 was scored as intermediate to

susceptible to all isolates and NC-D1 was scored as resistant to all isolates (Table 3).

However, at this time an allelism test has not been conducted. Zeller et al. (2002)

assigned Pm29 to the long arm of chromosome 7D. Since the gene in NC-D1 is on the

short arm of chromosome 7D, the Pm29 gene would be independent. The Pm29 gene was

derived from Ae. ovata and was shown to be independent of Pm19.

61

Spielmeyer et al. (2005) assigned Pm38 to chromosome 7DS in a T. aestivum

recombinant inbred line population. Pm38 is an adult plant resistance gene completely

associated with the Lr34/Yr18 gene(s). Liang et al. (2006) reported a quantitative trait

locus (QTL) for powdery mildew resistance in this same area in a ‘Fukuho-komugi’ x

‘Oligoculm’ population and concluded that the resistance was controlled by the

Lr34/Yr18 complex. The Fukuho-komugi linkage maps showed the QTL peak LOD

score was around the SSR marker Xgwm295. The SSR marker Xgwm295 is reported to

be about 25cM proximal to Xcfd66 on the wheat consensus map (Somers et al. 2004). In

our population, however, no genetic estimates or allele comparisons could be made

because Xgwm295 was null in both NC-D1 and Saluda. Lillemo et al. (2008) identified a

major QTL allele for powdery mildew resistance on chromosome 7DS in the cultivar

‘Saar’. They reported that the genetic factor controlling powdery mildew resistance was

located at the Lr34/Yr18 locus, and reported it to be 20cM proximal to Xcfd41 and

Xgdm88. Thus, both Liang et al. (2006) and Lillemo et al. (2008) positioned Pm38 at

least 40cM proximal to the Pm gene identifed in NC-D1 (Figure 1). Furthermore,

Lagudah et al. (2006) developed a sequence tagged site (STS) marker csLV34 that was

tightly linked and highly diagnostic for Lr34/Yr18. The csLV34 marker amplified a

fragment of 229 bp in both Saluda and NC-D1 which is characteristic of genotypes that

lack Lr34/Yr18/Pm38 (Lagudah et al. 2006). No other SSR markers proximal to cfd66

were polymorphic between NC-D1 and the recurrent parent Saluda, indicating there was

a terminal introgression terminating close to marker Xcfd66 in NC-D1. All together this

62

would confirm the Pm gene in NC-D1 is a novel gene on chromosome arm 7DS.

However, because an allelism test needs to be conducted between NC-D1 and Pm19, we

propose the resistance gene in NC-D1 should be temporarily designated MlNCD1.

The reported marker linkages and recessive nature of the flanking markers should

facilitate the incorporation of MlNCD1 into cultivar development programs. Because

marker Xgwm635 and Xcfd41 are linked to MlNCD1 in repulsion one generation of

selfing would be required to distinguish heterozygous marker carriers from homozygous

recurrent parental marker carries. Theses markers would be valuable when developing

Pm genes pyramids. BecauseMlNCD1 is located on chromosome 7DS, it can be readily

pyramided with other unlinked powdery mildew genes such as Pm25 (Shi et al. 1998),

Pm34 (Miranda et al. 2006), Pm 35 (Miranda et al. 2007), and Pm37 (Perugini et al.

2008) which are effective against natural powdery mildew populations in the eastern

United States (Parks et al. 2008). Because these Pm genes were identified in isolines with

the same adapted genetic background (Murphy et al., 1998, 1999a, 1999b, 2002), no non-

target marker selection would be required during pyramid building. Marker-assisted

selection would allow rapid development of adapted germplasm lines or advanced

breeding lines with multiple Pm genes. Pyramids of these Pm genes, not yet been

deployed commercially, would provide broad-spectrum resistance that should be more

durable than single race-specific resistance genes (Hsam and Zeller 2002).

63

Acknowledgments

We gratefully acknowledge Dr. Bob McIntosh for some excellent additions and

suggestions on the final draft of the manuscript. This research was supported by the

North Carolina Small Grain Growers Association.

64

References Bonnett DG, Rebetzke GJ, Spielmeyer W (2005) Strategies for efficient implantation of molecular markers in wheat breeding. Molecular Breeding 15:75-85 Bowen KL, Everts KL, Leath S (1991) Reduction in yield of winter wheat in North Carolina due to powdery mildew and leaf rust. Phytopathology 81:503-511 Cox TS, Raupp WJ, Wilson DL, Gill BS, Leath S, Bockus WW, Browder LE (1992) Resistance to foliar disease in a collection of Triticum tauschii germplasm. Plant Dis 76:1061-1064 Endo TR, Gill BS (1996) The deletion stocks of common wheat. J Hered 87:295-307 Everts KL, and Leath S (1992) Effect of early season powdery mildew on development, survival, and yield contribution of tillers of winter wheat. Phytopathology 82:1273-1278 Everts KL, Leath S, Finney PL (2001) Impact of powdery mildew and leaf rust on milling and baking quality of soft winter wheat. Plant Dis 85:423-429 Hardwick NV, Jenkins JEE, Collins B, Groves SJ (1994) Powdery mildew (Erysiphe graminis) on winter wheat: control with fungicides and the effects on yield. Crop Prot 13:93-98 Hsam SLK, Zeller FJ (2002), Breeding for powdery mildew resistance in common wheat (Triticum aestivum L.). In: Bélanger, RR., Bushnell WR, Dik AJ, and Carver TLW (eds) The Powdery Mildews: A Comprehensive Treatise. APS Press, St. Paul, MN. Huang XQ, Roder MS (2004) Molecular mapping of powdery mildew resistance genes in wheat: a review. Euphytica 137:203-223 Lagudah ES, McFadden H, Singh RP, Huerta-Espino J, Bariana HS, Spielmeyer W (2006) Molecular genetic characterization of the Lr34/Yr18 slow rusting resistance gene region in wheat. Theor Appl Genet 114:21-30 Leath S, Bowen KL (1989) Effects of powdery mildew, Triadimenol seed treatment, and Triadimefon foliar sprays on yield of winter wheat in North Carolina. Phytopathology 79:152-155 Leath S, Heun M (1990) Identification of powdery mildew resistance genes in cultivars of soft red winter wheat. Plant Dis 74:747-752 Liang SS, Suenaga K, He ZH, Wang ZL, Lui HY, Wang DS, Singh RP, Sourdille P, Xia XC (2006) Quantitative trait loci mapping for adult-plant resistance to powdery mildew in bread wheat. Phytopathology 96:784-789 Lillemo M, Asalf B, Singh RP, Huerta-Espino J, Chen XM, He ZH, Bjornstad A (2008) The adult plant rust resistance loci Lr34/Yr18 and Lr46/Yr29 are important determinants of partial resistance to powdery mildew in bread wheat line Saar. Theor Appl Genet (online)

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Lincoln SE, Daly MJ, Lander ES (1993) Constructing linkage maps with MAPMAKER/Exp Version 3.0. A tutorial reference manual, 3rd edn. Whitehead Institute for Medical Res., Cambridge Liu J, Liu D, Tao W, Li W, Wang S, Chen P, Cheng S, Gao D (2000) Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat. Plant Breed 119:21-24 Lutz J, Hsam LK, Limpert E, Zeller F (1994) Powdery mildew resistance in Aegilops tauschii Cross. and synthetic hexaploid wheats. Genet Res Crop Evol 41:151-158 Lutz J, Hsam LK, Limpert E, Zeller F (1995) Chromosomal location of powdery mildew resistance genes in Triticum aestivum L. (common wheat). 2. Genes Pm2 and Pm19 from Aegilops squarrosa L. Heredity 74:152-156 McIntosh RA, Hart GE, Devos KM, Gale MD, Rogers WJ, Dubcovsky J, Morris (2005) Catalogue of gene symbols for wheat: Annual supplement. http://wheat.pw.usda.gov/ggpages/wgc/2005upd.html. Cited 20 May 2008 McIntosh RA, Hart GE, Devos KM, Gale MD, Rogers WJ, Dubcovsky J, Morris (2006) Catalogue of gene symbols for wheat: Annual supplement. http://wheat.pw.usda.gov/ggpages/wgc/2006upd.html. Cited 20 May 2008 McIntosh RA, Hart GE, Devos KM, Gale MD, Rogers WJ, Dubcovsky J, Morris (2007) Catalogue of gene symbols for wheat: Annual supplement. http://wheat.pw.usda.gov/ggpages/wgc/2007upd.html. Cited 20 May 2008 Michelmore RW, Paran I, Kesseli VR (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88:9828-9832 Miranda LM, Murphy JP, Marshall D, Cowger C, Leath S (2007) Chromosomal location of Pm35, a novel Aegilops tauschii derived powdery mildew resistance gene introgressed into common wheat (Triticum aestivum L.). Theor Appl Genet 114:1451-1456 Miranda LM, Murphy JP, Marshall D, Leath S (2006) Pm34: a new powdery mildew resistance gene transferred from Aegilops tauschii Coss. to common wheat (Triticum aestivum L.). Theor Appl Genet 113:1497-1504 Mujeeb-Kazi A, Rajaram S (2002) Transferring alien genes from related species and genera for wheat improvement. In: Curtis BC, Rajaram S, Macpherson HG (eds) Bread wheat improvement and production. Food and Agriculture Organization of the United Nations, Rome, pp 199-215

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Murphy JP, Leath S, Huynh D, Navarro RA, Shi A (1998) Registration of NC96BGTD1, NC96BGTD2, and NC96BGTD3 wheat germplasm resistant to powdery mildew. Crop Sci 38:570-571 Murphy JP, Leath S, Huynh D, Navarro RA, Shi A (1999a) Registration of NC96BGTA4, NC96BGT45, and NC96BGTA6 wheat germplasm. Crop Sci. 39:883 Murphy JP, Leath S, Huynh D, Navarro RA, Shi A (1999b) Registration of NC97BGTD7 and NCBGTD8 wheat germplasms resistant to powdery mildew. Crop Sci. 39:883-884 Murphy JP, Navarro RA, Leath S (2002) Registration of NC99BGTAG11 wheat germplasm resistant to powdery mildew. Crop Sci. 42:1383 Niewoehner AS, Leath S (1998) Virulence of Blumeria graminis f. sp. tritici on winter wheat in the Eastern United States. Plant Dis 82:64-68 Parks R, Carbone I, Murphy JP, Marshall D, Cowger C (2008) Virulence structure of the Eastern U.S. wheat powdery mildew population. Plant Dis. 92:1074-1082 Perugini LD, Murphy JP, Marshall D, Brown-Guedira G (2008) Pm37, a new broadly effective powdery mildew resistance gene from Triticum timopheevii. Theor Appl Genet 116:417-425 Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233-234 Shi AN, Leath S, Murphy JP (1998) A major gene for powdery mildew resistance transferred to common wheat from wild einkorn wheat. Phytopathology 88:144-147 Snedecor GW, Cochran WG (1956) Statistical methods applied to experiments in agriculture and biology. The Iowa State College Press, Ames, IA Somers DJ, Isaac P, Edwards K (2004) A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor Appl Genet 109:1105-1114 Sourdille P, Singh S, Cadalen T, Brown-Guedira GL, Gay G, Qi L, Gill BS, Dufour P, Murigneux A, Bernard M (2004) Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (Triticum aestivum L.). Funct Integr Genomics 4:12-25 Spielmeyer W, McIntosh RA, Kolmer J, Lagudah ES (2005) Powdery mildew resistance and Lr34/Yr18 genes for durable resistance to leaf and stripe rust cosegregate at a locus on the short arm of chromosome 7D of wheat. Theor Appl Genet 111:731-735 Spielmeyer W, Singh RP, McFadden H, Wellings CR, Huerta-Espino J, Kong X, Appls R, Lagudah ES (2008) Fine scale genetic and physical mapping using interstitial deletion mutants of

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Lr34/Yr18: a disease resistance locus effective against multiple pathogens in wheat. Theor Appl Genet 116:481-490 Starling TM, Roane CW, Camper HM (1986) Registration of ‘Saluda’ wheat. Crop Sci 26:200 Stein N, Herren G, Keller B (2001) A new extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breed 120:354-356 Shtienberg D (1992) Effects of foliar dieases on gas exchange processes: A comparative study. Phytopathology 82:760-765 Tosa Y, Sakai K (1990) The genetics of resistance of hexaploid wheat to the wheatgrass powdery mildew fungus. Genome 33:225-230 Weisz R (2000) Small grain production guide 2000-2001. North Carolina Coop. Ext. Ser., Raleigh, NC Zeller FJ, Kong L, Hartl L, Mohler V, Hsam SLK (2002) Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.) 7. Gene Pm29 in line Pova. Euphytica 123:187-194 Zhou R, Zhu Z, Kong X, Huo N, Tian Q, Li P, Jin C, Dong Y, Jia J (2005) Development of wheat

near-isogenic lines for powdery mildew resistance. Theor Appl Genet 110:640-648

68

Table 2.1 Segregation ratios for powdery mildew reaction of F3 families from the NC-D1 /

Saluda wheat population in greenhouse and field evaluations.

Test Number of Families 

   χ21:2:1  P‐value 

   Resistant  Segregating  Susceptible       

Greenhouse  24  52  24  0.16  0.92 

Field  48  104  41  1.67  0.43 

69

Table 2.2 Segregation ratios for SSR markers among F2 individuals in the NC-D1/Saluda wheat population

Dominant marker  Co‐dominant marker SSR marker   AAa  Not AAb  AA  Hc  BBd  Total  χ2(1:3 or 1:2:1)  P‐value Xwmc506  55  131    186  2.07  0.15 Xgwm635  55  129    184  2.35  0.13 Xcfd41  40  133  173  0.33  0.57 Xgdm145  39  145  184  1.42  0.23 Xgdm88  33  96  51  180  4.40  0.11 Xcfd66  39  92  52  183  1.85  0.40 a AA = homozygous for the NC-D1 allele

b Not AA = heterozygous and homozygous for the Saluda allele

c H = heterozygous

d BB = homozygous for the Saluda allele

70

Table 2.3 Detached leaf reactions of NC-D1, XX186, Saluda, and the susceptible check

Chancellor to five powdery mildew isolates

Line / Cultivar 

Pm gene Blumeria graminis isolate 

Arapahoe  Asosan  E3‐14  Flat7‐11  85063 

NC‐D1  MlNCD1  Ra  R  R  R  R XX186  Pm19  S  S  S  I  S Saluda  Pm3a  S  S  I  S  S 

Chancellor  None  S  S  S  S  S 

R = Resistant; I = Intermediate; S = Susceptible

Figur

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71

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72

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73

Chapter III

MlAB10: a Triticum turgidum derived powdery mildew resistance gene identified in

common wheat

74

Abstract

Powdery mildew is an economically important disease in wheat growing areas

with a cool maritime environment. Genetic host resistance is the most economic,

consistent, and environmentally sound method of control. The wheat germplasm line

NC97BGTAB10 was genetically characterized as a source of a dominant race-specific

resistance gene. The F2-derived F3 population, developed from the cross

NC97BGTAB10 / Saluda, was evaluated in the field and greenhouse for its reaction to

powdery mildew. The population segregated in an expected 1:2:1 (resistant : segregating

: susceptible) ratio, typical of a monogenic trait. A bulked segregant analysis using 25 F3

families per bulk was conducted using the Diversity Array Technology (DArT). The

DArT analysis showed the most significant polymorphism between the bulks was on

chromosome 2BL. The population was then genotyped with SSR markers that were

specific to chromosome 2B and a linkage map was developed for this region. The closest

SSR marker, Xwmc445, mapped 7 cM proximal to the resistance gene and no marker was

mapped to the distal side of this gene. Xwmc445, mapped to the most distal deletion bin

(0.89-1.00) of chromosome 2BL. The powdery mildew resistance genes Pm6, Pm33 and

MlZec1 have also been mapped to chromosome 2BL. Both field reaction and linkage

analysis suggested that the resistance gene in NC97BGTAB10 was different than Pm6.

No allelism test could be conducted to determine the relationship between Pm33 or

MlZec1 and the resistance gene in NC97BGTAB10 at this time. Thus, this gene should

temporarily be designated MlAB10.

75

Introduction

Powdery mildew, caused by Blumeria graminis (syn. Erysiphe graminis) f. sp.

tritici, is an economically important disease of wheat (Triticum aestivum L.) in cool

maritime environments. Average grain yield losses due to powdery mildew epidemics

are reported to be around 34% on susceptible cultivars (Niewoehner and Leath 1998).

Reduced tiller survival, kernels per head, and kernel weight contribute to the yield

reduction (Bowen et al. 1991). In addition, decreased photosynthesis and transpiration

reduce the carbohydrate reserves available for grain fill (Shtienberg 1992). Together

these effects can negatively impact end-use quality parameters such as flour yield, protein

content, and softness equivalent (Everts et al. 2001).

Control methods for powdery mildew include chemical application, cultural

practices and the use of genetic resistance. Chemical and cultural control methods can be

detrimental to yield, erratic in effectiveness, and expensive (Hardwick et al. 1994; Leath

and Bowen 1989). Thus, genetic control has been pursued as the most consistent and

environmentally sound control method. Race-specific or qualitative resistance is the

most commonly used type of genetic resistance followed by adult-plant-resistance (APR)

or quantitative resistance (Huang and Roder 2004). Currently, 57 powdery mildew genes

at 41 loci have been formally designated (Huang and Roder 2004; McIntosh et al. 2005,

2006, 2007, personal communication).

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Disease surveys of the Eastern United States have shown no single race-specific

resistance gene has remained effective for long periods of time. Because of the continual

shifts towards higher virulence frequencies and an increased number of virulence gene

combinations in a fit background, powdery mildew populations have overcome widely

deployed resistance genes in a short period of time (Leath and Heun 1990; Niewoehner

and Leath 1997; Parks et al. 2008). Thus, it is imperative that new sources of powdery

mildew resistance are identified and incorporated into adapted genetic backgrounds.

More than half of the Pm genes have been introgressed from diploid and

tetraploid relatives of wheat (Huang and Roder 2004). Wild emmer wheat, Triticum

turgidum var. dicoccoides (AABB; 2n = 28), one of the wild progenitors of common

wheat, has been reported to be a valuable source of disease resistance genes (Xie and

Nevo 2008). Since the A and B genomes of emmer wheat and common wheat are

homologous, simple breeding procedures enable direct gene transfer through homologous

recombination between the two species (Mujeeb-Kazi and Rajaram 2002). Powdery

mildew resistance genes Pm26 (Rong et al. 2000), Pm30 (Liu et al. 2002), Pm33 (Zhu et

al 2005), and Pm36 (Blanco et al. 2008), as well as temporarily designated genes MlG

(Xie et al. 2003), MlZec1 (Mohler et al. 2005), and MlIW72 (Ji et al. 2008), were all

introgressed into common wheat from T. dicoccoides.

The use of PCR based molecular markers to tag genomic regions of interest has

become routine in wheat improvement. The identification of molecular markers flanking

disease resistance genes simplifies breeding activities such as cultivar development

77

(Bonnett et al. 2005), near isogenic line development (Zhou et al., 2005), and pyramiding

resistance genes into single genotypes by marker assisted selection. Many of the recently

reported Pm genes have markers associated with them (Miranda et al. 2006, 2007;

Perugini et al. 2008). Gene pyramiding has been shown to have an enhanced level of

resistance when compared to single genes (Lui et al. 2000; Barloy et al. 2007; Kloppers

and Pretorius 1997). Theory suggests pyramids should provide more durable resistance

(Hsam and Zeller 2002).

The germplasm line NC97BGTAB10 was released by the North Carolina State

University Small Grains breeding program in 1997. It continues to exhibit a high level of

resistance to naturally occurring field isolates of powdery mildew in the Eastern United

States. The objectives of this research were to genetically characterize the powdery

mildew resistance in NC97BGTAB10, identify SSR markers closely linked to the

resistance gene, and provide evidence that the gene is a novel T. dicoccoides-derived

powdery mildew resistance gene.

78

Material and methods

Plant Material

The powdery mildew resistant germplasm line NC97BGTAB10 (PI 604036) was

crossed with the susceptible cultivar Saluda (PI 480474). NC97BGTAB10, hereafter

referred to as NC-AB10, is an F5-derived line with the pedigree Saluda*3//’Ward’/PI

471746 (Navarro et al. 2000). PI 471746 is winter growth habit wild emmer accession

collected in central Israel. Ward (CI 15892 is a spring-sown durum wheat developed and

released by North Dakota State University. Saluda is a soft red winter wheat developed

and released by Virginia Polytechnic Institute and State University (Starling et al. 1986).

Saluda possesses the powdery mildew resistance gene Pm3a, which is not effective

against naturally occurring powdery mildew populations in the Southeastern United

States (Parks et al. 2008). The NC-AB10 / Saluda F1 hybrid produced the 139 F2 seeds,

which were used to produce the 139 F3 lines for greenhouse and field evaluations.

Reserve F2 seed was saved and used to determine the gene action of the powdery mildew

resistance gene in NC-AB10.

79

Greenhouse evaluations

One hundred and thirty-nine F2:3 lines plus parents were evaluated for their

reactions to powdery mildew during 2007 and 2008. An experimental unit was two 10-

cm pots each containing five seedlings per genotype under evaluation. The parental lines

NC-AB10 and Saluda were included at 10-pot intervals as controls. Parental seeds were

derived from selfed progenies of the original plants used to produce the populations

under study. A randomized complete block design was used where replications occurred

over time, i.e. replication one was during 2007 and replication 2 occurred in 2008. The

greenhouse was maintained at 24oC/20oC (day/night), and the plentiful natural light was

supplemented with artificial High Intensity Discharge 1000W lights. An additional 100

reserve F2 plants from the cross NC-AB10 / Saluda were grown in Ray Leach “Cone-

tainers” (Stuewe and Sons, Inc.) under the same greenhouse conditions to determine the

gene action of the powdery mildew resistance gene in NC-AB10. Ten seeds of each NC-

AB10 and Saluda were included as checks.

The plants were inoculated at Feekes growth stage 2 to 3 by gently shaking

conidia from leaves of infected Saluda plants onto the leaves of the F2:3 lines and control

plants. The inoculation source was a powdery mildew isolate ‘Yuma’, which is

maintained by the USDA-ARS plant pathology laboratory at North Carolina State

University. Disease evaluations were made 10 to 15 days post inoculation, or when

Saluda showed intense and uniform signs of powdery mildew infection across the whole

80

experiment. Disease severity scores were on a scale from 0 to 9, as described by Srnic et

al. (2005). Briefly, 0-3 = resistant, immune with no visible signs of infection to

detectable amount of mycelium growth; 4-6 = intermediate, mycelium and conidial

production increased from slight to moderate; 7-9 = susceptible, with increasing amount,

size, and density of mycelium and conidia to a fully compatible reaction. The F2:3 lines

with similar disease reaction to NC-AB10 were classified as ‘resistant’ and those similar

to Saluda were classified as ‘susceptible’. Lines that exhibited both resistant and

susceptible plants were classified as ‘segregating’. Chi-square tests were conducted to

test the goodness of fit between observed and expected segregation ratios (Snedecor and

Cochran, 1956).

Field evaluation

The same 139 F2:3 lines evaluated in the greenhouse were also evaluated at the

Cunningham Research and Education Center at Kinston, NC in October 2007. The

experimental design was a randomized complete block with two replications. The

experimental unit was comprised of a 1.2m row planted with 60 to 70 seeds. NC-AB10

and Saluda were included as controls every 40 plots. Twenty-eight other wheat

differential lines with known Pm genes were included in each replication. These lines

contained the Pm genes Pm1a, Pm1c, Pm2, Pm3a, Pm3b, Pm3c, Pm3d, Pm3f, Pm3g,

Pm4a, Pm4b, Pm5a, Pm5b, Pm5d, Pm6, Pm7, Pm8, Pm9, Pm12, Pm16, Pm17, Pm20,

81

Pm21, Pm25, Pm34, Pm35, Pm37 (Table 1). A four row border of Saluda surrounded the

experiment to promote a powdery mildew epidemic. Irrigation, fertilization, and other

agronomic practices were conducted as needed following standard management practices

for North Carolina (Weisz 2000).

Disease severity was recorded during April and May of 2008 when all Saluda

rows showed uniform signs of powdery mildew infections. Plants were evaluated at

Feekes growth stage 9 to 10.1. Disease severity was recorded on 20 to 30 plants per plot

using the same 0 to 9 scale as the greenhouse evaluation. Lines that had a mean disease

severity score of 0-3 and a disease phenotype resembling that of the NC-AB10 were

scored as ‘resistant’, while those with a severity score of 7-9 and resembling that of

Saluda were scored as ‘susceptible’. Families with a mix of resistant and susceptible

plants were scored as segregating. A chi-square test was conducted to test the goodness

of fit between observed and expected segregation ratios (Snedecor and Cochran 1956).

Pearson’s correlation coefficient (r) was used to test the relationship between field and

greenhouse data (Steel et al. 1997). The least square difference (LSD) was calculated

using the Proc GLM procedure in SAS (SAS 2002).

Marker analysis

The CTAB procedure described by Stein et al. (2001) was used to extract

genomic DNA from leaf tissue of the 139 F2 plants that gave rise to the F2:3 lines by.

82

DNA from 25 homozygous resistant and 25 homozygous susceptible lines was pooled for

bulked segregant analysis with the Diversity Arrays Technology (DArT) platform by

Triticarte Pty Ltd (Wenzl et al. 2007). Genome and chromosome specific markers were

used to genotype the 139 individuals to develop a linkage map of the NC-AB10

introgression. Wheat SSR primers were synthesized according to sequence published in

the GrainGenes database (http://wheat.pw.usda.gov), with all forward primers modified

to include the M13 sequence (CACGACGTTGTAAAACGAC-) at the 5’ end for labeling

purposes (Schulke 2000). PCR reaction conditions and visualization were conducted as

described by Miranda et al (2006). The sequence tagged site (STS) marker

NAU/STSBCD135-1 was evaluated on ten resistant, ten susceptible, and parental lines to test

for the presence of Pm6. Reactions and visualization for NAU/STSBCD135-1 were

performed according to Ji et al. (2008). Chinese Spring (CS) aneuploid and deletion

stocks, kindly provided by the Wheat Genetic and Genomic Resources Center at Kansas

State University, were used to confirm the chromosomal location of the linked SSR

markers. The CS stocks included the nullisomic 2B-tetrasomic 2D (N2BT2D),

nullisomic 2D-tetrasomic 2B (N2DT2B), ditelosomic 2BL (Dt2BL), deletion line 2BL-3

(Fraction Length (FL) = 0.35), deletion line 2BL-5 (FL = 0.59), deletion line 2BL-10 (FL

= 0.69) and deletion line 2BL-6 (FL = 0.89) (Endo and Gill 1996). The CS Ditelosomic

2BS (Dt2BS) (LPGKU2009, E04-Dt2BS-2) was obtained from the National BioResource

Project-Wheat, Japan (http://www.nbrp.jp/report/reportProject.jsp?project=wheat).

83

Chi-squared analyses were used to test for expected segregation of markers and

linkage analysis was conducted with MAPMAKER/EXP (version 3.0b) (Lincoln et al.

1993). The Kosambi mapping function was used to estimate centimorgan (cM) distances

between the markers. Maximum likelihood estimates confirmed map orders of the wheat

consensus map (Somers et al. 2004).

84

Results

Greenhouse and field evaluations

NC-AB10 and Saluda had mean disease severity scores of 0.5 and 7.4,

respectively, in the greenhouse evaluations. The observed segregation for the NC-AB10 /

Saluda F2:3 population fit a 1:2:1 (resistant:segregating:susceptible) ratio, which is

expected for a monogenic trait (Table 1). NC-AB10 and Saluda had mean disease

severity scores of 1.3 and 7.5, respectively, in the field evaluation. The observed

segregation in the field also fit the expected segregation ratio for monogenic inheritance

of the powdery mildew resistance present in NC-AB10 (Table 1). NC-AB10 expressed a

high level of resistance in the field evaluation when compared to the differential wheat

lines with known Pm genes (Table 2).

Marker analysis

Triticarte Pty Ltd used 214 DArT markers to test for polymorphism between the

resistant and susceptible bulks and parental lines. Seventeen markers had a significant

allele frequency difference between the bulks and parental lines (data not shown). Of

those 17 DArT markers, markers wPt-4892 and wPt292 were reported on the distal tip of

chromosome 2BL (Mantovani et al. 2008), one was located on chromosome 7A, and 13

85

had an unknown location. The sequences of the most significant DArT markers were

unavailable to develop a PCR based marker at this time (Wenzl personal

communication).

Wheat SSR markers specific for chromosomes 7A and 2B were used to determine

the chromosome location of the resistance gene. Seven SSR markers specific to

chromosome 7A were genotyped on 10 resistant and 10 susceptible lines. No significant

associations were detected between the markers and the powdery mildew reaction. Five

SSR markers located on the distal arm of chromosome 2BL (Somers et al 2004) were

polymorphic between the resistant and susceptible bulks and NC-AB10 and Saluda,

respectively. Linkage analysis of the mapping population located markers Xwmc445,

Xwmc317, Xwmc149, Xwmc361, Xwmc817, and Xwmc526 7 cM, 15 cM, 18 cM, 18cM,

25 cM, and 37 cM proximal to the powdery mildew resistance gene present in NC-AB10,

respectively (Figure 1). Markers Xwmc445, Xwmc149, and Xwmc317 were dominant

markers that produced bands of 270bp, 243bp, and 155bp in Saluda and were null in NC-

AB10. Markers Xwmc361 and Xwmc817 were co-dominant markers that produced bands

of 236bp and 149bp in Saluda and 259bp and 142bp in NC-AB10, respectively. Marker

Xgwm526 was a dominant marker which produced a 167bp band in NC-AB10 and was

null in Saluda. All SSR markers proximal to Xwmc526 in the wheat consensus map

(Somers et al. 2004) and the International Triticeae Mapping Initiative (ITMI) map (Xue

et al. 2008) were non-polymorphic between NC-AB10 and Saluda. All mapped markers

did not significantly deviate from their expected segregation ratios (Table 3).

86

All the linked SSR markers were reported to map at more than one location,

except Xwmc361 and Xwmc317 (Somers et al. 2004; Xue et al. 2008). The CS aneuploid

stocks were used to confirm that the markers segregating in the NC-AB10 / Saluda

population were located on the long arm of chromosome 2B. The results showed that the

fragments associated with the resistance in NC-AB10 for markers Xwmc445 (270bp), and

Xwmc526 (167bp) were present in CS (173bp and 246bp, respectively), Dt2BL, and

N2DT2B, but absent in Dt2BS and N2BT2D (Figure 2). This confirmed that the

chromosome location of these markers is most likely chromosome 2B. In addition, these

markers were deletion-mapped using four CS deletion stocks. The markers did not

produce the fragments associated with the resistance in any of the deletion lines (Figure

2). This confirms that the most likely position of the markers linked to the resistance

gene in NC-AB10, and of the gene itself, is the most distal deletion bin (0.89-1.00) of

chromosome 2BL.

87

Discussion

NC-AB10 exhibited a high level of powdery mildew resistance in both the

greenhouse and field evaluations. Segregation ratios from both evaluations confirmed

that the powdery mildew resistance in NC-AB10 is controlled by a single gene. NC-

AB10 has been stable throughout all field evaluations in North Carolina since it was

released in 1997 and would be an effective parent if used in a breeding program.

There are three designated genes and one temporary-designated gene

characterized on chromosome 2B. The recessive gene Pm26, a T. dicoccoides-derived

powdery mildew gene from accession TTD140, is located to the short arm of

chromosome 2B (Rong et al. 2000). The gene Pm6 was identified in the wheat line TP

114, which was derived from a T. timopheevi derivative, and characterized by Jorgensen

and Jensen (1973). Pm6 has been mapped on the chromosome 2BL with the closest

RFLP marker reported as Xbcd135 (Tao et al. 2000). The RFLP marker was mapped by

Sourdille et al (2004) to the deletion bin centromere-2BL-2(0-0.36). This would place

Pm6 in the middle to the proximal end of chromosome 2BL. In addition Pm6 is not

effective against the powdery mildew field isolates in North Carolina (Table 2), thus it is

assumed that the resistance gene in NC-AB10 is different than Pm6.

The Pm33 gene was identified in the T. carthlicum accession PS5 by Zhu et al.

(2005). Pm33 was reported to be on the distal portion of chromosome 2BL, between

markers Xgwm526 and Xwmc317, at a genetic distance of 18.1 cM and 1.1 cM,

88

respectively. This location appears to be about 15 cM proximal to the NC-AB10 gene in

the present study. Further, the marker Xgwm526 was co-dominant with a PCR product of

160bp associated with the Pm33 resistance gene. In contrast, Xgwm526 was a dominant

marker with a PCR product of 165bp and was associated with the susceptible allele in the

NC-AB10 population. A similar comparison can be made with Xwmc317. It was a

dominant marker with a PCR product of 123bp associated with resistance in the Pm33

study, whereas it produced a PCR product of 155bp and was associated with the resistant

parent in the NC-AB10 population. Pm33 and the gene in NC-AB10 are derived from

different subspecies of T. turgidum, and appear to map to different although closely

linked regions of 2BL. Seed of the T. carthlicum accession PS5 was unavailable and no

allelism test could be conducted at this time. Thus, we were unable to determine if the

resistance gene in NC-AB10 was novel or an allele of Pm33. Finally, the temporary-

designated MlZec1 was mapped to the most distal deletion bin (2BL-6 0.89-1) by Mohler

et al. (2005). The MlZec1 gene was identified from a T. dicoccoides line Zecoi-1 and

was reported to be linked to the distal side Xgwm526 and Xwmc356 at 14.2 cM and 2 cM,

respectively. A detached leaf test indicated that the resistance gene in NC-AB10 and

MlZec1 are different; however, no allelism test has been conducted to determine their

relationship. Therefore, we propose this T. diccoccoidies-derived dominant gene be

temporarily designated MlAB10.

Wild relatives of wheat are valuable sources of novel and useful disease resistance

genes. Because powdery mildew populations are in a continual state of change though

89

migration and sexual recombination, they are persistent in overcoming effective race-

specific resistance genes in wheat (Parks et al. 2008). The most recent notable reduction

in effective powdery mildew control has been Pm17. In a powdery mildew disease

survey across the eastern US, there was a dramatic increase in virulence to Pm17 between

2003 and 2005 (Parks et al. 2008). This shows that race-specific resistance genes can be

overcome in as little as three years in widely deployed genes. Gene pyramids are

hypothesized to have a more broad-spectrum and durable resistance (Hsam and Zeller

2002). Thus, marker assisted selection (MAS) could be implemented to efficiently

pyramid the resistance gene reported herein with other recently reported Pm genes that

are in an adapted background, such as Pm34, Pm35, Pm37, and MlNCD1 (Miranda et al.

2006, 2007; Perugini et al. 2007; Maxwell et al. 2008).

90

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Shtienberg D (1992) Effects of foliar dieases on gas exchange processes: A comparative study. Phytopathology 82:760-765 Snedecor GW, Cochran WG (1956) Statistical methods applied to experiments in agriculture and biology. The Iowa State College Press, Ames, IA Somers DJ, Isaac P, Edwards K (2004) A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor Appl Genet 109:1105-1114 Sourdille P, Singh S, Cadalen T, Brown-Guedira GL, Gay G, Qi L, Gill BS, Dufour P, Murigneux A, Bernard M (2004) Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (Triticum aestivum L.). Funct Integr Genomics 4:12-25 Srnic G, Murphy JP, Lyerly JH, Leath S, Marshall DS (2005) Inheritance and chromosomal assignment of powdery mildew resistance genes in two winter wheat germplasm lines. Crop Sci 45:1578-1586 Steel RGD, Torrie JH, Dickey DA (1997) Principles and procedures of statistics and biometrical approach, 3rd edn. McGraw-Hill, New York, New York, pp286-290 Stein N, Herren G, Keller B (2001) A new extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breed 120:354-356 Tao W, Liu D, Liu J, Feng Y, Chen P (2000) Genetic mapping of the powdery mildew resistance gene Pm6 in wheat by RFLP analysis. Theor Appl Genet 100:564-568 Weisz R (2000) Small grain production guide 2000-2001. North Carolina Coop. Ext. Ser., Raleigh, NC Wenzl P, Raman H, Wang J, Zhou M, Huttner E, Kilian A (2007) A DArT platform for quantitative bulked segregant analysis. BMC Genomics 8:196-205 Xie C, Sun Q, Ni Z, Yang T, Nevo E, Fahima T (2003) Chromosomal location of a Triticum dicoccoides-derived powdery mildew resistance gene in common wheat by using microsatellite markers. Theor Appl Genet 106:341-345 Xie W, Nevo E (2008) Wild emmer: genetic resources, gene mapping and potential for wheat improvenment. Euphytica (online) Xue S, Zhang Z, Lin F, Kong Z, Cao Y, Li C, Yi H, Mei M, Zhu H, Wu J, Xu H, Zhao D, Tian D, Zhang C, Ma Z (2008) A high-density intervarietal map of the wheat genome

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enriched with markers derived from expressed sequence tags. Theor Appl Genet 117:181-189 Zhu Z, Zhou R, Kong X, Dong Y, Jia J (2005) Microsatellite markers linked to 2 powdery mildew resistance genes introgressed from Triticum carthlicum accession PS5 into common wheat. Genome 48: 585-590 Zhou R, Zhu Z, Kong X, Huo N, Tian Q, Li P, Jin C, Dong Y, Jia J (2005) Development of wheat near-isogenic lines for powdery mildew resistance. Theor Appl Genet 110:640-648

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Table 3.1 Segregation ratios for powdery mildew reaction of F2:3 lines from the NC-

AB10 / Saluda wheat population evaluated in two replications in both greenhouse and

field tests.

Test Number of F2:3 lines χ2 (1:2:1) P-value Resistant Segregating Susceptible Greenhouse 40 72 27 2.61 0.27 Field 37 70 25 2.67 0.26

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Table 3.2 Powdery mildew reactions of differential wheat lines and NC-AB10 under

infection with natural inoculum in the field evaluation in 2008.

Differential Line Pm gene Chromosome

Location Severity Score Reaction Type

CI 14114 1a 7AL 1.0 R PI 351489 1c 7AL 4.0 I CI 14118 2 5DS 7.5 S CI 14120 3a 1AS 7.0 S CI 14121 3b 1AS 7.0 S CI 15886 3c 1AS 8.0 S W150 3e 1AS 3.0 R C6815-Pm3f 3f 1AS 8.0 S Aristide 3g 1AS 6.5 S CI 14123 4a 2AL 7.5 S RONOS 4b 2AL 4.2 I CI 14125 5a 7BL 6.5 S Kormoran 5b 7BL 6.0 S I5 5d 7BL 7.0 S CI 15888 6 2BL 5.0 I Transec 7 4BS.4BL-2RL 6.5 S Kavkaz 8 1BL.1RS 7.0 S N14 9 7A 5.0 I Wembly 12 6BS 1.5 R Pm16 16 4A 3.5 R Amigo 17 1AL.1RS 5.5 I GHSE-Pm20 20 6BS.6RL 4.5 I DH2 21 6VS.6AL 1.0 R NC96BGTA5 25 1AS 1.5 R NC97BGTD7 34 5DL 0.5 R NC96BGTD3 35 5DL 0.5 R NC96BGTAG11 37 7AL 0.5 R NC-AB10 MlAB10 2BL 0.5 R Saluda 3a 1AS 7.5 S Chancellor none - 8.0 S LSD 3.1

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Table 3.3 Segregation ratios for SSR markers among F2 individuals in the NC-AB10 / Saluda wheat population

SSR

Marker Dominant marker Co-dominant marker Total χ2(1:2:1 or

3:1) P-

value AA Not AA Not BB BB AA AB BB Xwmc149 35 103 138 0.01 0.92 Xwmc317 35 103 138 0.01 0.92 Xwmc445 36 88 124 1.08 0.30 Xgwm526 104 31 135 0.30 0.58 Xwmc361 35 65 37 137 0.42 0.81 Xwmc817 37 70 30 137 0.78 0.68

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Figure 3.1 Mapping position of MlAB10 on chromosome 2BL. SSR marker names are to

the right and the estimated distance in centimorgans is to the left.

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Figure 3.2 Gel image of the CS anueploid and deletion stocks used to confirm the

location of the linked SSR markers Xgwm526 (A) and Xwmc445 (B). Lanes 1-11

represent NC-AB10, Saluda, Chinese Spring, N2BT2D, N2DT2B, Dt2BL, Dt2BS, del

2BL-3, del 2BL-5, del 2BL-10, del 2BL-6, respectively.

A

B

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Chapter IV

Identification and Genetic Mapping of Two Triticum timopheevii-Derived Powdery

Mildew Resistance Genes in Common Wheat

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Abstract

Wheat powdery mildew is an economically important disease in cool and humid

environments. Powdery mildew causes yield losses as high as 48 percent through a

reduction in tiller survival, kernels per head and kernel size. Race-specific host

resistance is the most consistent, environmentally friendly and economical method of

control. The wheat germplasm lines NC06BGTAG12 and NC06BGTAG13 possess

novel genetic resistance to powdery mildew. Phenotypic evaluation of F2:3 families

derived from the crosses NC06BGTAG12 / Jagger and NC06BGTAG13 / Jagger

indicated that the resistance in both germplasm lines was controlled by a single dominant

gene. Phenotypic evaluation of 224 F2:3 families from a cross between NC06BGTAG12

and NC06BGTAG13 suggested that the resistant genes in each germplasm were allelic,

because all families were resistant. Bulk segregant analysis revealed that SSR markers

specific for the 7AL chromosome segregated with the resistance genes. Linkage

mapping found the SSR markers Xwmc273 and Xwmc346 mapped 7.2 cM distal and 2.4

cM proximal, respectively, in NC06BGTAG12 / Jagger, while they mapped 8.3 cM distal

and 6.6 cM proximal, respectively, in NC06BGTAG13 / Jagger. The multiallelic Pm1

locus maps to this region of chromosome 7AL. A detached leaf test with differential

powdery mildew isolates indicated that the resistance genes in NC06BGTAG12 and

NC06BGTAG13 were different from each other and different from all the alleles at the

Pm1 locus. Additional allelism tests need to be conducted with the germplasm lines and

the Pm1 locus, but the genes in NC06BGTAG12 and NC06BGTAG13 have been given

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the temporary designation MlAG12 and MlAG13.

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Introduction

Powdery mildew, caused by Blumeria graminis (syn. Erysiphe graminis) DC. f.

sp. tritici Em. Marchal, is an economically important disease of wheat in growing regions

with cool and humid environments. This biotrophic fungus reduces grain yields by as

much as 48% on susceptible cultivars during severe epidemics (Everts and Leath 1992).

Powdery mildew infections have negative impacts on end-use quality parameters of

wheat caused by the depletion of carbohydrate reserves available for grain fill (Shtienber

1992). Powdery mildew can be observed on susceptible cultivars from the seedling stage

through head emergence and causes a decrease in number and survival of tillers, kernels

per head, and kernel weight (Leath and Bowen 1989).

Host resistance in the form of race-specific, monogenic, or qualitative resistance

is the optimal type of control for powdery mildew in wheat. Race-specific host resistance

is the most cost effective, environmentally sound and consistent method of control

(Hardwick et al.1994; Leath and Bowen 1989). Currently 57 powdery mildew resistance

(Pm) genes at 40 loci have been formally designated (Huang and Röder 2004; McIntosh

et al. 2005, 2006, 2007, personal communication). However, due to the rapid defeat of

widely deployed Pm genes and an increase in the mean number of virulent genes

possessed by powdery mildew populations, the effectiveness of race-specific resistance

genes is ephemeral (Niewoehner and Leath 1997; Parks et al 2008).

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Breeding for powdery mildew resistance has relied heavily on the primary and

secondary wheat gene pools for new sources of resistance. Triticum timopheevii (Zhuk.)

Zhuk. subsp armeniacum (Jakubz.) van Slageren (2n=2x=28; genome AAGG) is a

valuable resource for powdery mildew resistance. To date, three resistance genes, Pm6

(Jorgensen and Jensen 1973), Pm27 (Peusha et al. 2000), and Pm37 (Perugini et al.

2008), have been transferred from T. timopheevii into common hexaploid wheat.

The long arm of chromosome 7A is a key genomic region for powdery mildew

resistance. The Pm1 locus with five alleles (a-e) is located on 7AL. Alleles at the Pm1

locus trace to T. monococcum, T. spelta, and T. aestivum (Hsam et al. 1998; Singrun et al

2003). Additional powdery mildew genes, Mlm2033, Mlm80, and MlIW72 have been

mapped to the Pm1 region and were identified in T. monococcum and T. dicoccoides,

respectively (Ji et al, 2008; Yao et al. 2007). Additional resistance genes including Pm9,

mlRD30, and PmU have all been mapped in close proximity to the Pm1 locus on

chromosome arm 7AL (Schneider et al. 1991; Singrun et al. 2004; Qui et al. 2005).

The identification of molecular markers closely linked to powdery mildew

resistance genes is common (Huang and Röder 2004, Miranda et al. 2006, 2007; Perugini

et al. 2008). Molecular markers facilitate the pyramiding of resistance genes into inbred

lines or cultivars to increase the durability of the race-specific resistance (Liu et al. 2000;

Hsam and Zeller 2002). Marker-assisted selection (MAS) can decrease the breeding

population size and increase the frequency of favorable alleles at the target loci under

selection (Bonnett et al. 2005). Many different marker systems have been employed on

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powdery mildew resistance genes (see Huang and Röder 2004 for a review), but

microsatellite or simple sequence repeat (SSR) markers are the most polymorphic and

cost effective marker system identified thus far in wheat.

The identification of new and novel sources of powdery mildew resistance in

adapted germplasm is beneficial for wheat breeders in the Southeastern United States.

Further identification of molecular markers linked to each new source of powdery

mildew resistance would be advantageous for MAS in cultivar development. The

objectives of this study were to determine the inheritance, chromosomal location, and

linkage of molecular markers to genes for resistance to powdery mildew in the two

germplasm lines NC06BGTAG12 and NC06BGTAG13 and to determine whether the

resistance genes were novel.

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Materials and Methods

Plant materials

Two powdery mildew resistant wheat germplasm lines, NC06BGTAG12 (NC-

AG12) and NC06BGTAG13 (NC-AG13), were each crossed with the powdery mildew

susceptible hard red winter wheat Jagger (PI 593688). NC-AG12 (PI 642416) is a

BC2F7-derived line with the pedigree Saluda*3 / PI 538457 (Murphy et al. 2007). PI

538457 is a powdery mildew resistant T. timopheevii subsp. armeniacum accession

collected in Iraq. NC-AG13 (PI 642417) is a BC2F6-derived line with the pedigree

Saluda*3 / PI 427442. PI 427442 is a powdery mildew resistant T. timopheevii subsp.

armeniacum accession collected in Iraq. Saluda (PI 480474) is a soft red winter wheat

developed and released by Virginia Polytechnic Institute and State University (Starling et

al. 1986). Saluda possesses the powdery mildew resistance gene Pm3a, which is

ineffective against the naturally occurring powdery mildew population in North Carolina

(Parks et al. 2008). One hundred twenty eight random F2:3 families were developed from

the NC-AG12 / Jagger cross and 136 random F2:3 families were developed from the NC-

AG13 / Jagger cross. An additional population developed from the cross NC-AG12 /

NC-AG13 comprised 224 random F2:3 families and was utilized to test for allelism

between the germplasm lines. Four other F2 populations derived from the crosses NC-

AG12 / Saluda, NC-AG13 / Saluda, NC-AG12 / Axminster and NC-AG13 / Axminster

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were developed. The NC-AG12 / Saluda and NC-AG13 / Saluda were used to determine

the gene action of the powdery mildew genes. Axminster (PI 228307) is a powdery

mildew resistant T. aestivum cultivar that possesses the gene Pm1a. The populations NC-

AG12 / Axminster and NC-AG13 / Axminster were used to determine the genetic

relationship of the resistance genes in NC-AG12 and NC-AG13 to the Pm1 locus.

Phenotypic evaluation

a) Detached leaf evaluations

Lines possessing Pm1a, Pm1b, Pm1c, Pm1d, and Pm1e along with NC-AG12,

NC-AG13, Saluda, and the powdery mildew susceptible check Chancellor (CI 12333)

were evaluated in a detached leaf test as described by Perugini et al. (2008). Ten

different powdery mildew isolates collected from wheat fields in North Carolina were

used for the evaluation. The isolates were chosen based on their ability to differentiate

between the Pm1 alleles and the germplasm lines NC-AG12 and NC-AG13 (Cowger

personal communication).

b) Whole plant evaluations

All F2:3 lines, from the NC-AG13 / Jagger and the NC-AG12 / Jagger populations,

as well as parental lines were evaluated for their reactions to powdery mildew during the

2006 - 2007 winter in the greenhouse. An experimental unit was two 10-cm pots each

containing five seedlings per genotype under evaluation. The parental lines NC-AG12 or

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NC-AG13 and Jagger were included at 10-pot intervals as controls. Parental seeds were

derived from selfed progenies of the original plants used to produce the populations

under study. A randomized complete block design with one replication, where pot pairs

were randomly assigned a position in the greenhouse in both years. The greenhouse was

maintained at 24oC/20oC (day/night), and provided plentiful natural light supplemented

with artificial High Intensity Discharge 1000W lights.

The four F2 populations were plant in Ray Leach “Cone-tainers” (Stuewe and

Sons, Inc.) and grown under the same greenhouse conditions as described above. Tens

seeds of each parental lines where included as checks in the evaluations.

For all greenhouse evaluations, plants were inoculated at Feekes growth stage 2 to

3 by gently shaking conidia from leaves of infected Saluda plants onto the leaves of the

F2:3 lines and control plants. The inoculation source was a powdery mildew isolate

‘Yuma’, which is maintained by the USDA-ARS plant pathology laboratory at North

Carolina State University. Disease evaluations were made 10 to 15 days post inoculation,

or when Saluda showed intense and uniform signs of powdery mildew infection across

the whole experiment. Disease severity scores were on a scale from 0 to 9 as described

by Srnić et al. (2005). Briefly, 0-3 = resistant, immune, no visible signs of infection to a

detectable amount of mycelium growth; 4-6 = intermediate, mycelium and conidial

production increased from slight to moderate; 7-9 = susceptible, with increasing amount,

size, and density of mycelium and conidia to a fully compatible reaction. The F2:3 lines

with similar disease reaction to NC-AG12 or NC-AG13 were classified as ‘resistant’ and

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those similar to Jagger were classified as ‘susceptible’. Lines with both resistant and

susceptible plants were classified as ‘segregating’. Chi-square tests were conducted to

test the goodness of fit between observed and expected segregation ratios (Snedecor and

Cochran, 1956).

All 224 F2:3 families in the NC-AG12 / NC-AG13 population were evaluated in

the greenhouse in 2008 as described above, with two replicates in time. In addition, a

random subset of 138 families was evaluated in the field during the 2007 – 2008 growing

season at the Cunningham Research and Education Center at Kinston, NC. The field

experimental design was a randomized complete block with two replications. The

experimental unit was comprised of a 1.2m row planted with 60 to 70 seeds. NC-AG12,

NC-AG13, and Saluda were included as controls every 40 plots. Twenty-eight additional

germplasm lines with known Pm genes were included in each replication. A four row

border of Saluda surrounded the experiment to promote a powdery mildew epidemic.

Irrigation, fertilization, and other agronomic practices were conducted as needed

following standard management practices for North Carolina (Weisz 2000). Disease

severity ratings were recorded during April to May of 2008 or when the Saluda plots

showed uniform signs of powdery mildew infection. Disease severity scoring was

followed as in the greenhouse test (described above).

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Marker analysis

Genomic DNA was extracted from leaf tissue of the F2 plants that gave rise to the

F2:3 families by the CTAB procedure described by Stein et al. (2001). DNA from 10

homozygous resistant and 10 homozygous susceptible lines was utilized for a modified

bulked segregant analysis (BSA) with chromosome and genome specific SSR markers

(Michelmore et al. 1991; Röder et al.1998; Somers et al. 2004; Xue et al. 2008). The

individuals selected for the modified BSA were not bulked together, but were genotyped

individually. Markers that were polymorphic between the resistant / susceptible classes,

parental germplasm lines NC-AG12 and NC-AG13, Jagger, and Saluda were identified as

linked to the resistance gene and subsequently used to genotype the populations. All the

individuals, from their respective populations, were genotyped with SSR markers to

develop a linkage map around the resistance genes indentified in NC-AG12 and NC-

AG13. Amplification and visualization of the SSR markers was conducted according to

Miranda et al. (2006). Wheat SSR primers were synthesized according to sequence

published in the GrainGenes database (http://wheat.pw.usda.gov), with all forward

primers modified to include the M13 sequence (CACGACGTTGTAAAACGAC-) at the

5’ end for labeling purposes (Schulke 2000).

Chi-squared analyses were used to test for expected segregation of markers and

linkage analysis was conducted with MAPMAKER/EXP (version 3.0b) (Lincoln et al.

1993). The Kosambi mapping function was used to estimate centimorgan (cM) distances

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between the markers. Maximum likelihood estimates confirmed map orders of the wheat

consensus map (Somers et al. 2004).

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Results

Inheritance of powdery mildew resistance

NC-AG12 and NC-AG13 had mean disease severity scores of 0.0 and 0.5,

respectively, while Jagger had a mean severity score of 9 in the greenhouse evaluations.

Field disease severity scores for NC-AG12, NC-AG13 and Jagger were 1.0, 2.0, and 8.0

respectively. Of the 128 F2:3 lines tested from the NC-AG12 / Jagger population, 32 were

homozygous resistant, 73 were segregating, and 23 were homozygous susceptible,

indicating monogenic control of the resistance in NC-AG12 (Table 1). Of the 136 F2:3

lines tested from the NC-AG13 / Jagger population, 23 were homozygous resistant, 79

were segregating, and 34 were homozygous susceptible, indicating monogenic control of

the resistance in NC-AG13 (Table 1). For the cross NC-AG12 / Saluda, 128 F2 plants

were evaluated and 26 were scored as susceptible to 99 scored as resistant. Similar

results were observed in the NC-AGH13 / Saluda populations, where 30 F2 plants were

scored as susceptible to 94 scored as resistant. Both populations, the NC-AG12 / Saluda

and NC-AG13 /Saluda, segregated in an expected ratio of three resistant to on susceptible

(χ2 = 1.67 and 0.04 respectively; P-value = 0.28 and 0.84 respectively), which indicates

the resistance was controlled by a dominate gene. Of the 138 and 224 F2:3 families from

the NC-AG12 / NC-AG13 population evaluated in the field and greenhouse, respectively,

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all families were scored as resistant, suggesting that the genes in NC-AG12 and NC-

AG13 were allelic (Table 1).

Molecular Mapping

Twelve wheat SSR markers that mapped to the A and B genomes were used to

screen 10 homozygous resistant and 10 homozygous susceptible individuals from each

population. One marker, Xwmc525, was polymorphic between the two bulks and the

parental lines. Xwmc525 mapped to the long arm of chromosome 7A on the wheat

consensus map (Somers et al. 2004). An additional nine markers specific to chromosome

7AL were tested and found to be polymorphic between the individual classes and

parental lines. Of the markers that segregated with the resistance genes, Xwmc346,

Xwmc525, Xcfa2040, and Xwmc116 were all co-dominant markers. Markers Xgwm332,

Xwmc273, and Xwmc809 were dominant markers linked in repulsion with the resistance

alleles. Finally, markers Xwmc790 and Xmag2185 were dominant markers linked in

coupling with the resistance allele. All the markers that were placed on the linkage map

were test on Saluda to determine the most likely interval for the T. timopheevii

introgression. The markers Xwmc346, Xwmc525, Xwmc273, Xwmc116, and Xcfa2040

were polymorphic between the germplasm lines (NC-AG12 and NC-AG13) and Saluda,

indicating that the introgression was delimited .by Xcfa2040 and Xwmc273.

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All nine markers were genotyped on 128 and 136 individuals from the NC-AG12

/ Jagger and NC-AG13 / Jagger populations, respectively. Linkage analysis showed that

the resistance genes in NC-AG12 and NC-AG13 were flanked by markers Xwmc346 and

Xwmc273 at distances of 6.6 cM and 8.3 cM, respectively in NC-AG12, and 2.4 cM and

7.2 cM, respectively in NC-AG13 (Figure 1). All markers included in the linkage map

did not deviate from their expected ratio. All the markers used in this study were

previously mapped to the terminal deletion bin (0.86-1.00) of chromosome 7AL

(Sourdille et al. 2004; Ji et al. 2008; Perugini et al. 2008).

Detached leaf test

Differential reactions were observed on lines that possessed Pm1a, Pm1b, Pm1c,

Pm1d, Pm1c, Jagger, Chancellor, Saluda, NC-AG12, and NC-AG13 when inoculated

with 5 powdery mildew isolates (Table 3). The NC-AG12 and NC-AG13 were

differentiated from each other as well as all the Pm1 alleles. These results combined with

the genetic mapping results indicated that the powdery mildew resistance genes present in

NC-AG12 and NC-AG13 are novel and are alleles at the Pm1 locus or closely linked to

the Pm1 locus. Further allelism tests need to be conducted in order to determine whether

the powdery mildew resistance genes reported herein are alleles of the Pm1 locus or are

just tightly linked to the Pm1 locus. However, from these experimental results the

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authors suggest the T. timopheevii-derived resistance genes present in NC-AG12 and NC-

AG13 be temporarily designated MlAG12 and MlAG13, respectively.

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Discussion

One effective dominant powdery mildew resistance gene was identified in both

NC-AG12 and NC-AG13. The resistance genes MlAG12 and MlAG13 were derived

from the tetraploid relative T. timopheevii and have been transferred into a hexaploid

wheat background that is adapted to the southeastern United States. Both genes mapped

to the long arm of chromosome 7AL with the most likely interval between the SSR

markers Xwmc273 and Xwmc346. Both NC-AG12 and NC-AG13 have shown consistent

and broad spectrum resistance in the field (data not shown) and would be good candidates

for use in a breeding program.

Wheat chromosome 7AL is a hot spot for powdery mildew resistance genes. The

genes Pm1, Pm9, Pm37, mlRD30, PmU, Mlm2033, Mlm80, MlW72, as well as two

unnamed genes derived from T. monococcum, have all been located to the 7AL

chromosome. Of all the loci identified on chromosome 7AL, the Pm1 locus is the most

complex with multiple alleles that confer dominant powdery mildew resistance and

include Pm1a, Pm1b, Pm1c, Pm1d, and Pm1e (Hsam et al. 1998). Of the alleles at the

Pm1 locus, Pm1a, Pm1c, and Pm1e were identified in a T. aestivum background (Sears

and Briggle 1969; Hsam et al. 1998; Singrum et al. 2003). The alleles Pm1c and Pm1d

were introgressed from T. monoccoccum and T. spelta var. duhamelianum, respectively

(Hsam et al. 1998). The Pm1 locus mapped close to the SSR marker Xgwm344, which

placed the locus in the terminal deletion bin (0.86-1.00) of chromosome 7AL (Ji et al.

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2008; Singrun et al. 2003; Perugini et al. 2008). Pm37, a T. timopheevii-derived gene,

was located about 16.2 cM proximal to the Pm1 locus (Perugini et al. 2008). MlAG12

and MlAG13 do not appear to be alleles of the Pm37 locus, since the markers flanking

Pm37, Xwmc790 and Xgwm332, were not polymorphic between the parental germplasm

lines and Saluda. This would indicate that the T. timopheevii introgressions in NC-AG12

and NC-AG13 are distal to the introgession for Pm37. In addition, Xwmc790 mapped

about 34 cM and 48.6 cM proximal to MlAG12 and MlAG13 respectively.

The recessive resistance gene Pm9 was identified in the spring wheat cultivar

Normandie and mapped 8.5 cM distal to the Pm1 locus (Schneider et al. 1991). The Pm9

gene is ineffective in North Carolina and exhibited a stark difference to both NC-AG12

and NC-AG13 by having a disease severity reaction of 5 comapred to 0.5 for both NC-

AG12 and NC-AG13. Another recessive resistance gene mlRD30, identified in the

common wheat line TA2682a, mapped 1.8 cM distal to the Pm1 locus or marker

Xgwm344 (Singrun et al. 2004). Because MlAG12 and MlAG13 are dominant, neither

would appear to be allelic to either the Pm9 or mlRD30 genes. The T. urartu-derived

resistance gene PmU mapped to a similar region on chromosome 7AL located 2.2 cM

distal to the SSR marker Xwmc273 (Qui et al. 2005). Two other T. monococcum-derived

resistance genes Mlm2033 and Mlm80 were fine-mapped and found to be tightly linked to

the marker Xmag2185 at less than 1 cM (Yao et al. 2007). The marker Xmag2185

mapped in the same region as the Pm1 locus or the marker Xgwm344. Most recently the

T. dicoccoides-derived resistance gene MlIW72 mapped at 3.3 cM proximal to the marker

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Xmag2185 or 4.9 cM proximal to Xgwm344. Taken together, these reports suggest that

the region flanking the Pm1 locus is a hot spot for disease resistance genes. However,

because of the close proximity of the markers, it remains unclear whether many of these

genes form a resistance gene cluster or are alleles of each other.

The identification and introgression of novel powdery mildew resistance genes,

and the identification of molecular markers linked to these genes, are key to consistent

genetic control of wheat powdery mildew. Powdery mildew virulence surveys in the

eastern US have shown that the virulence structure of the powdery mildew population

continues to evolve. More complex pathogen virulence patterns have been identified that

overcome widely deployed resistance genes (Niewoehner and Leath 1998; Parks et al.

2008). Host gene pyramiding is a common breeding strategy that should result in more

effective genetic control. Marker assisted selection is a key component of this approach

because it is effective in identification of genotypes with multiple genes. Phenotypic

selection of genotypes with gene pyramids can be very difficult. Not only will marker

assisted selection facilitate gene pyramiding, but it can also reduce the amount of non-

target genetic background (Bonnett et al. 2005). This study provides new sources of

adapted powdery mildew resistance for use in wheat breeding programs in the

southeastern US. The two genes reported herein have the potential to be pyramided with

powdery mildew genes at other loci, such as Pm34, Pm35, which have been introgressed

into a similar genetic background (Miranda et al. 2006, 2007). Further molecular

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markers could be used to link MlAG12 or MlAG13 in coupling with the genes Pm37 or

Pm9, which could then also be used in a cultivar development program.

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Acknowledgments

This research was supported, in part, by the North Carolina Small Grain Growers

Association.

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Table 4.1 Segregation ratios for powdery mildew reaction of F2:3 families from the NC-

AG12 x Jagger (NC-AG12), NC-AG13 x Jagger (NC-AG13), and NC-AG12 x NC-

AG13 crosses

Population Experiment Segregation χ2 P-value Resistant Segregating Susceptible NC-AG12 Greenhouse 32 73 23 3.8a 0.15 NC-AG13 Greenhouse 23 79 34 5.34a 0.07

NC-AG12 x NCAG13

Greenhouse 224 0 0 177b < 0.01 Field 138 0 0 288b < 0.01

a Chi-square was calculated based on an expected segregation of 1:2:1

(Resistant:Segregating:Susceptible)

b Chi-square was calculated based on an expected segregation of 7:8:1

(Resistant:Segregating:Susceptible)

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Table 4.2 Segregation ratios for the SSR markers that flank the powdery mildew

resistance genes in NC-AG12 and NC-AG13

Population Xwmc346 Xwmc273 AAa Hb BBc χ2 P-Value AA H/BBd χ2 P-value

NC-AG12 29 78 32 2.21 0.33 31 101 0.16 0.69 NC-AG13 32 94 48 4.07 0.13 42 126 0.79 0.37

a AA homozygous for the NC-AG12 or NC-AG13 allele

b H heterozygous

c BB Homozygous for the Jagger allele

d H/BB not homozygous for the NC-AG12 or NC-AG13 allele (dominant marker for the

Jagger allele)

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Table 4.3 Reaction of six genotypes possessing Pm1 alleles, NC-AG12, NC-AG13, Saluda, Jagger, and the susceptible control

Chancellor after infection with 5 isolates of Blumeria graminis f. sp. tritici (Bgt)

Cultivar/ Line Pm gene

Bgt isolates Arapahoe Asosan E314 Flat

7-11 85063 101a2 #8 127 ABK Yuma

Axminster Pm1a Ra R S R R S I S S R MocZlatka Pm1b R R S R R I R S S R M1N Pm1c S S R S S S S I I R T. spelta Pm1d R R S R R I R I I R Ovest Pm1e S S S I S S S S S S NC-AG12 MlAG12 R R R R R R R R R R NC-AG13 MlAG13 R R I R R R R R I R Saluda Pm3a S S I S S R R S S S Jagger None S S S S S S S S S S Chancellor None S S S S S S S S S S

a R = resistant; I = intermediate; S = susceptible

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Figure 4.1 Map positions of the wheat powdery mildew resistance genes in NC-AG12

(A) and NC-AG13 (B) on chromosome 7AL. The dotted arrow indicates the direction

toward the centromere. Marker names are to the right and the distances between markers

in centimorgans (cM) are to the left.