IMPROVING DISEASE RESISTANCE TO STEM RUST AND POWDERY MILDEW IN
WHEAT USING D GENOME INTROGRESSIONS FROM AEGILOPS TAUSCHII
By
Andrew Thomas Wiersma

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Plant Breeding, Genetics and Biotechnology - Crop and Soil Sciences - Doctor of Philosophy
2017

i

ABSTRACT
IMPROVING DISEASE RESISTANCE TO STEM RUST AND POWDERY MILDEW IN
WHEAT USING D GENOME INTROGRESSIONS FROM AEGILOPS TAUSCHII
By
Andrew Thomas Wiersma
Stem rust (Puccinia graminis) and powdery mildew (Blumeria graminis) are persistent threats to
hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD) production worldwide. Genetic
variation for disease resistance has been limited in the D genome of wheat due to restricted gene
flow between T. aestivum and the diploid D genome progenitor species, Aegilops tauschii Coss.
(2n=2x=14, DD). One method to introgress disease resistance from Ae. tauschii to wheat is
through direct hybridization and backcrossing with wheat. Using this method, the Ug99-effective
stem rust resistance gene SrTA10187 was previously introgressed from Ae. tauschii accession
TA10187 and mapped to wheat chromosome 6DS. Development of a high-resolution genetic
map surrounding SrTA10187 assigned the resistance locus to a 1.1 cM interval on 6DS and
enabled candidate gene identification. To introgress and map powdery mildew resistance in the
D genome, introgression lines (ILs) were developed by direct hybridization of the resistant Ae.
tauschii accession TA1662 with the susceptible wheat line KS05HW14. Following embryo
rescue and recurrent backcrossing to KS05HW14, ILs were developed that only segregate for D
genome alleles. Using a combination of genotyping-by-sequencing and KASP™ SNP markers, a
novel powdery mildew resistance gene, designated Pm58, was mapped to 2DS and confirmed to
be effective under field conditions. Powdery mildew resistant germplasm, fixed for Pm58 were
released for immediate use in disease resistance breeding.

ii

Copyright by
ANDREW THOMAS WIERSMA
2017

iii

To Kailyn, with love

iv

ACKNOWLEDGEMENTS
First and foremost, I would like to thank my Ph.D. advisor, Dr. Eric Olson, for his mentorship
throughout my graduate education and research. He showed me firsthand the art and science of
plant breeding.

I would also like to thank my graduate committee members Dr. C. Robin Buell, Dr. Mitch
McGrath, and Dr. Dean DellaPenna for their time, support, and guidance. Dr. C. Robin Buell
gave me the best introduction to plant bioinformatics and Michigan State University that I could
ever imagine. Dr. Mitch McGrath taught me the importance of population development and
germplasm improvement. Dr. Dean DellaPenna taught my favorite class out of 23 years of
education, and always knew the right questions to ask.

I would like to thank my M.S. degree advisors, Dr. Phil Westra and Dr. Jan Leach, for equipping
me for continued graduate education.

I would like to thank my fellow graduate student colleagues for their continuous support and
friendship—especially Ben Mansfield and Linda Brown.

Finally, I would like to thank the Michigan Wheat Program and the Monsanto Company for
supporting my research and graduate education in plant breeding.

v

TABLE OF CONTENTS
LIST OF TABLES ....................................................................................................................... ix
LIST OF FIGURES .......................................................................................................................x
CHAPTER I ...................................................................................................................................1
Introduction ....................................................................................................................................1
Global importance of wheat ..............................................................................................2
The origins of hexaploid wheat .........................................................................................2
Aegilops tauschii .................................................................................................................3
Biotrophic wheat pathogens ..............................................................................................4
Stem rust...................................................................................................................4
Powdery mildew .......................................................................................................6
Disease management and resistance breeding in wheat .................................................7
Integrated disease management ...............................................................................7
Major gene and quantitative resistance ...................................................................8
Adult plant resistance and Mlo genes ......................................................................9
The importance of continued disease resistance breeding ....................................10
Accessing D genome genetic variation for disease resistance ......................................11
Limited D genome genetic variation ......................................................................11
Synthetic hexaploid wheat......................................................................................12
Direct hybridization ...............................................................................................14
Advancements in genetic and genomic tools for wheat ................................................15
Genome sequencing ...............................................................................................15
Wheat genotyping platforms ..................................................................................16
Genotyping-by-sequencing ....................................................................................16
KASP™ Markers ...................................................................................................17
Problem definition ...........................................................................................................18
Objectives..........................................................................................................................19
REFERENCES .................................................................................................................20
CHAPTER II ................................................................................................................................29
Fine mapping of the stem rust resistance gene SrTA10187 ......................................................29
CHAPTER III ..............................................................................................................................30
Identification of Pm58 from Aegilops tauschii ...........................................................................30
CHAPTER IV...............................................................................................................................31
Registration of Two Wheat Germplasm Lines Fixed for Pm58 ..............................................31
Abstract .............................................................................................................................32
Introduction ......................................................................................................................32
Methods .............................................................................................................................33
Germplasm line development .................................................................................33
Powdery mildew evaluation ...................................................................................34
vi

Agronomic evaluation ............................................................................................35
Characteristics..................................................................................................................35
Powdery mildew resistance....................................................................................35
Agronomic evaluations ..........................................................................................37
Discussion..........................................................................................................................37
Availability........................................................................................................................39
Acknowledgements ..........................................................................................................40
REFERENCES .................................................................................................................41
CHAPTER V ................................................................................................................................43
Conclusions and future perspectives ..........................................................................................43
Overview of dissertation research ..................................................................................44
Overcoming past limitations in disease resistance breeding ........................................46
Future perspectives ..........................................................................................................47
APPENDIX ...................................................................................................................................48
REFERENCES .............................................................................................................................61

vii

LIST OF TABLES
Table 4.1 Isolate-specific powdery mildew reactions identified in wheat lines U6714-A-011,
U6714-B-056, KS05HW14, and the Bgt susceptible check Jagalene .......................................36
Table 4.2 Grain yield LS-means of locally adapted check varieties, KS05HW14, U6714-A011, and U6714-B-056 in 10 diverse environments throughout the United States ................38
Table 5.1 Segregation of Pst resistance in BC2F4-derived introgression lines and BC3F2 and
F2 mapping populations...............................................................................................................55
Table 5.2 Seedling and adult Pst infection types and severity on wheat lines KS05HW14,
U6719-004, and U6719-009..........................................................................................................56
Table 5.3 Grain yield LS-means of locally adapted check varieties, KS05HW14, U6719-004,
and U6719-009 in 10 diverse environments throughout the United States ............................57

viii

LIST OF FIGURES
Figure 1.1 Photographs of common barberry (Berberis vulgaris) identified in the Manistee
National Forest of Michigan .........................................................................................................5
Figure 1.2 Diagrams of A) synthetic hexaploid wheat development and B) direct
hybridization between Ae. tauschii and hexaploid wheat ........................................................13
Figure 5.1 Introgression of stripe rust resistance from Ae. tauschii accession TA1718 and
mapping population development ..............................................................................................51
Figure 5.2 Pstv-37 seedling leaf infection types of KS05HW14, TA1718, Ambassador, and
resistant and susceptible BC3F2 and F2 individuals ..................................................................59

ix

CHAPTER I
Introduction

1

Global importance of wheat
The historical, cultural, and economic significance of wheat is immense. The shift from
nomadic- to agrarian-based human societies coincided with the domestication of wheat, and
wheat has remained a cornerstone of ancient and modern civilizations (Shewry 2009). Presently,
wheat accounts for approximately 20% of the calories consumed by humans worldwide
(Brenchley et al. 2012). The success and wide-adoption of wheat can be attributed, in part, to
grain storage proteins that form a gluten matrix. Gluten is an important source of protein and is
responsible for the large diversity of wheat-based food products including bread, noodles, cake,
pastries, and cereal (Shewry 2009). The genetic diversity and plasticity of wheat has also enabled
rapid adaptation of wheat to various growing environments including Asia, Europe, the
Americas, and Africa while maintaining high grain yield. Globally, annual production of wheat
surpasses 700 million tonnes—95% of which is common wheat (Triticum aestivum L.) and the
remaining 5% is durum wheat (Triticum turgidum L. subsp. durum Desf.) (FAOSTAT 2014,
Dubcovsky and Dvorak 2007).

The origins of hexaploid wheat
The earliest forms of cultivated wheat include the diploid species Triticum monococcum
L. (2n = 2x = AA) and the tetraploid species T. turgidum subsp. dicoccoides L. (2n = 4x =
AABB) commonly known as einkorn and emmer wheat, respectively. These wheat species were
domesticated from natural populations and likely maintained as landraces by early farmers
(Feldman 2005). Approximately 9,000 to 10,000 years ago, the natural hybridization between T.
turgidum and the diploid wild goat grass species, Aegilops tauschii (2n = 2x = DD), led to the
speciation of modern allohexaploid wheat, T. aestivum (2n = 6x = AABBDD) (Kihara 1944,

2

McFadden and Sears 1946). Based on the limited divergence between the D genomes of Ae.
tauschii and T. aestivum, most agree that the natural hybridization between T. turgidum and Ae.
tauschii occurred very few times or only once in history (Cox 1997, Baidouri et al. 2017).
Through the continued cultivation of hexaploid wheat, non-shattering and free-threshing variants
were identified and preserved.

Aegilops tauschii
Extensive research has focused on the distribution, and genotypic and phenotypic
diversity of Ae. tauschii because of its crucial role as the D genome progenitor of wheat. The
native range of Ae. tauschii extends from Transcaucasia to central Asia between the 30°N and
45°N parallels (Pestsova et al. 2000). Using spike morphology, early studies classified Ae.
tauschii into two subspecies—subsp. tauschii and subsp. strangulata (Dvorak 1998). More
recent studies based on single nucleotide polymorphisms (SNPs) have classified Ae. tauschii into
two distinct lineages, L1 and L2, with very few intermediate types (Wang et al. 2013). Generally,
Ae. tauschii subpp. tauschii group within L1 and are acclimated to elevations higher than 400 m
above sea level, while Ae. tauschii spp. strangulate group within L2 and are acclimated to
elevations lower than 400 m above sea level (Mizuno et al. 2010, Wang 2013). Phylogenetic
analysis, of Ae. tauschii collections indicated that accessions most closely related to the D
genome of wheat belong to the L2E sublineage as described by Wang et al. (2013). Accessions
belonging to this sublineage originate from the southern coast of the Caspian Sea, which
supports the hypothesis that wheat was domesticated in that region (Wang et al. 2013).

3

Biotrophic wheat pathogens
Biotrophic pathogens cause many of the most destructive diseases affecting wheat. A few
of the most notable biotrophic pathogens of wheat include Puccinia triticina f.sp. tritici (leaf
rust), P. striiformis f.sp. tritici (stripe rust), P. graminis f.sp. tritici (stem rust), and Blumeria
graminis f.sp. tritici (powdery mildew). As obligate biotrophs, they each rely on living plant
tissue for growth and propagation. To remain undetected by the host, biotrophs use specialized
structures called haustoria to infect and interface with the plant cell to suppress host-defense and
obtain nutrients (Panstruga 2003). When conditions are favorable for disease development, each
of these pathogens has the potential to cause large-scale epidemics that can reduce grain yield
and cause economic and social hardship (McIntosh et al. 1995, Cowger et al. 2012).

Stem rust
Stem rust is a disease affecting wheat and other small grains that is caused by the fungal
basidiomycete Puccinia graminis f.sp. tritici. Stem rust can be observed on wheat as red-brown
(rust colored) oval lesions on leaves, leaf sheaths, and stems. When the infection is severe,
tearing of the epidermal layer of plant tissues and brittle stems leads to reduced photosynthetic
capacity and plant lodging (McIntosh et al. 1995). Puccinia graminis is a macrocyclic,
heteroecious rust with a life cycle involving five types of spores and two plant hosts. Starting
during the warm summer months, red-brown asexual urediniospores are produced on infected
wheat which can then reinfect surrounding wheat. As the wheat begins to senesce and autumn
approaches, black teliospores are produced that overwinter and undergo karyogamy and meiosis
to produce basidiospores. In the spring, basidiospores infect the alternative host, common

4

Figure 1.1 Photographs of common barberry (Berberis vulgaris) identified in the Manistee
National Forest of Michigan (Wiersma, 2016).

5

barberry (Berberis vulgaris L., Figure 1.1), where a dikaryotic mycelium is formed.
Pycniospores and aeciospores are produced on the adaxial and abaxial surfaces of barberry
leaves, respectively, and aeciospores are capable of infecting wheat—thereby completing the
cycle (Petersen 1974).
In the early 20th century, after repeated stem rust epidemics in the central Great Plains of
North America, and amidst rising fears about food security following World War I, a multi-state
barberry eradication program began that ultimately saw the removal of approximately 500
million plants (Peterson et a. 2005). In theory, by disrupting the reproductive life cycle of P.
graminis, the pathogen could no longer overwinter in northern states and eventually the disease
might be eliminated. Unfortunately, the extent to which windborne urediniospores could travel
had not yet been realized. The term “Puccinia Pathway” was coined to describe how asexual
urediniospores were capable of overwintering in the Gulf States and Mexico, and of traveling on
monsoon wind currents to northern states (Aylor 2003). While the barberry eradication did not
eliminate stem rust in northern states, it was effective at delaying the arrival of the disease. Also,
the number of stem rust races decreased and stabilized by reducing the sexual reproduction and
genetic recombination of P. graminis (Jin 2011).

Powdery mildew
Wheat powdery mildew is caused by the fungal ascomycete Blumeria graminis f.sp.
tritici, and appears as white-to-grey mycelium and conidia on wheat leaves, leaf sheaths, and
stems. As B. graminis matures, dark reproductive structures called cleistothecia can be seen
among the white mycelium. Characteristics making B. graminis particularly difficult to control
are its short reproductive cycle and rapid production of secondary inoculum (conidia) that can

6

germinate in high relative humidity (rather than requiring free water) (Te Beest et al. 2008).
Powdery mildew is most severe in intensively managed wheat production systems where the use
of nitrogen fertilizers, irrigation, and semi-dwarf wheat varieties lead to dense and compact
canopies, optimal for disease development. Powdery mildew is considered a cool season
pathogen because it favors temperatures between 10 and 22°C (Cowger et al. 2012). Generally,
powdery mildew has the greatest impact on wheat production in the northern hemisphere in
coastal regions or areas with high humidity and annual rainfall—including the eastern and midwest soft wheat growing regions of the United States (Niewoeher et al. 1998, Parks et al. 2008).

Disease management and resistance breeding in wheat
Integrated disease management
The most effective method to control biotrophic fungal diseases of wheat, including stem
rust and powdery mildew, is one that integrates cultural methods with the use of foliar fungicides
and genetic resistance. Common cultural methods used to minimize the risk of stem rust and
powdery mildew are wider plant or row spacing, optimized irrigation timing, rotation to non-host
crops, and removal of weeds, volunteer wheat, and alternative hosts (Schumann and D’Arcy
2012). Foliar fungicides such as strobilurin, triazole, and mixed mode of action fungicides can be
applied at critical growth stages to protect grain yield (Dimmock and Gooding 2002). Despite the
importance of cultural and chemical disease management, the most effective strategy also
includes genetic resistance.
Genetic resistance to biotrophic plant pathogens takes advantage of the multilayered plant
innate immunity. The primary line of defense against biotic threats is pattern-triggered immunity
(PTI), which involves recognition of non-adapted microbes and phytopathogens at the cell

7

surface by pattern recognition receptors (PRRs) such as receptor-like kinases and receptor-like
proteins (Nejat et al. 2016, Bautrot and Zipfel 2017). A secondary line of defense is effectortriggered immunity (ETI), which typically involves the indirect or direct recognition of pathogen
virulence factors (effectors) by intracellular nucleotide-binding/leucine-rich-repeat (NLR)
receptors (Cui et al. 2015). ETI leads to programmed cell death at the site of infection (termed
the hypersensitive reaction), which restricts the invading pathogen to a tissue dead-zone and
limits its growth and proliferation (Li et al. 2015).
Although many factors limit the effectiveness of genetic resistance, it remains the most
economically affordable and environmentally safe approach to reduce the risk of stem rust and
powdery mildew in wheat (Ellis et al. 2014, Acevedo-Garcia et al. 2017). Additionally, the
harvest index (grain weight/above ground biomass weight) of modern wheat is nearly optimized,
and additional yield gains will likely be made by protecting wheat photosynthetic tissue from
biotic and abiotic stresses, including biotrophic pathogens (Curtis and Halford 2014).

Major gene and quantitative resistance
There are two primary types of genetic resistance in wheat disease resistance breeding—
major gene (qualitative) and quantitative resistance. As the name suggests, major gene resistance
is controlled by a single locus in the wheat genome, whereas quantitative resistance is controlled
by many small effect loci (Poland et al. 2008). Major gene resistance is typically race-specific
and involves detection of the pathogen or signatures of pathogen attack by large and genetically
diverse families of NLR or PRR resistance genes (Petit-Houdenot and Fudal 2017). So far, all
the stem rust and powdery mildew major genes that have been cloned in wheat, Sr35, Sr33, Sr50,
Sr22, Sr45, Pm3b, and Pm2, belong to the NLR gene family (Saintenac et al. 2013, Periyannan et

8

al. 2013, Mago et al. 2015, Steuernagel et al. 2016, Yahiaoui et al. 2003, Sanchez-Martin et al.
2016). The immune response produced by major gene resistance create strong selection pressure
on the pathogen which can lead to increased frequency of virulent races that breakdown host
resistance. Alternatively, quantitative resistance is generally considered a more durable form of
resistance because it is polygenic and it reduces disease rather than providing complete immunity
(Brown 2015).

Adult plant resistance and Mlo genes
Other forms of resistance that do not fit neatly into the major gene or quantitative
resistance categories include adult plant resistance and the recessive mlo alleles of barley and
wheat (Li et al. 2014, Acevedo-Garcia et al. 2014). Adult plant resistance is described as a partial
reduction in disease that is not correlated with a seedling resistance phenotype. Adult plant
resistance is particularly useful to plant breeders because it is generally more durable than major
gene resistance, it often enhances major gene resistance, and it can be effective against a broad
range of races and pathogens (Ellis et al. 2014). One example of adult plant resistance in wheat is
the pleiotropic locus Lr34/Yr18/ Sr57/Pm38 which confers resistance to leaf rust, stripe rust
(yellow rust), stem rust, and powdery mildew, respectively. Map-based cloning of Lr34 revealed
that a transmembrane ABC transporter was solely responsible for multi-pathogen resistance
(Krattinger et al. 2009).
In barley, durable and broad-spectrum resistance to B. graminis f.sp. hordei is due to lost
function of the Mlo gene which codes for a transmembrane protein that is necessary for pathogen
penetration of the host-cell (Schulze-Lefert and Vogel 2000). While mlo genes could be a source
of powdery mildew resistance in hexaploid wheat, all three homoeologous Mlo genes must be

9

mutated to provide resistance. This was first demonstrated by Wang et al. (2014) using a
transcription activator-like effector nuclease (TALEN) to mutate a conserved region in each of
the homoeologous Mlo alleles. Recently, the same result was reproduced without the use of
transgenes. Acevedo-Garcia et al. (2017) screened a wheat TILLING population for missense
mutations in Mlo homoeologues and pyramided three mutant alleles into a single wheat line to
confer resistance.

The importance of continued disease resistance breeding
Breeding for genetic resistance to stem rust and powdery mildew remain key objectives
in wheat due to the rapid evolution of P. graminis and B. graminis. In 1999, a new stem rust race
with virulence to the widely used Sr31 resistance gene posed a significant threat to global wheat
production (Pretorius et al. 2000). What came to be known as the Ug99-race group spread from
Uganda to surrounding eastern African countries and the Middle East, and gained additional
virulence to Sr24 and Sr36 (Jin et al. 2008, Jin et al. 2009, Singh et al. 2011). More recently, in
2016, the highly virulent stem rust race TTTTF was discovered in Sicily and represents a major
threat to European wheat production (Bhattacharya 2017). Likewise, powdery mildew isolates
collected from the eastern soft wheat growing region of the United States have overcome most of
the major gene resistance available in commercial varieties (Parks et al. 2008). Due to the rapid
reproductive cycle of B. graminis, the Fungicide Resistance Action Committee has listed cereal
powdery mildew as having high risk of developing resistance to fungicides (www.frac.info, Dec
2013). If the limited number of fungicides available to control powdery mildew become
ineffective, wheat growers will be forced to rely even more heavily on genetic resistance.

10

Accessing D genome genetic variation for disease resistance
Limited D genome genetic variation
A great deal of effort has been focused on expanding the genetic variation in hexaploid
wheat, especially within the D genome. As demonstrated by the wide global distribution of bread
wheat, the acquisition of a D genome from Ae. tauschii enabled hexaploid wheat to be grown in
more diverse environments compared to tetraploid wheat (Dubcovsky and Dvorak 2007).
However, the genetic variation within the D genome of wheat is limited due to the domestication
bottleneck, or founder effect, which involved very few natural hybridizations between tetraploid
wheat and Ae. tauschii (Cox 1997). The comparatively large amount of genetic variation within
the Ae. tauschii gene pool has not been fully incorporated into cultivated and landrace varieties
of hexaploid wheat because gene flow is restricted between the species (Reif et al. 2005). Plant
breeders rely on the genetic diversity available within wheat to select for improved wheat traits
when confronted with diverse biotic and abiotic stresses. If the genetic variation within wheat is
insufficient to make genetic gain towards a breeding target, additional genetic variation can be
introduced from the wild relatives of wheat—especially Ae. tauschii (Feuillet et al. 2007). In
fact, Ae. tauschii is one of the most accessible wheat relatives because it belongs to the primary
gene pool of wheat and Ae. tauschii chromosomes can readily pair and recombine with the D
genome of wheat (Ogbonnaya et al. 2013).
Plant breeders implement various methods of interspecific and backcross hybridization
between wheat and its wild relatives to transfer desirable traits from Ae. tauschii to hexaploid
wheat (Cox 1997). The two best defined methods are the development of synthetic hexaploid
wheat and direct hybridization of hexaploid wheat with Ae. tauschii. To date, novel genetic
variance derived from Ae. tauschii has been used to improve a wide variety of traits in wheat,

11

including disease and insect resistance, yield components, and tolerance to abiotic stresses such
as precocious germination, drought, heat, boron, and aluminum (Borner et al. 2015). In 2011,
Rouse et al. screened a diverse collection of Ae. tauschii accessions and discovered that
approximately 22% of the accessions were resistant to Ug99 races of P. graminis. To date, six
stem rust resistance genes have been introgressed from Ae. tauschii to wheat: Sr33, Sr45, Sr46,
SrTA1662, SrTA10171, and SrTA10187 (Periyannan et al. 2013, Periyannan et al. 2014, Yu et al.
2015, Olson et al. 2013a, Olson et al. 2013b). Likewise, Ae. tauschii has been used for
introgression of powdery mildew resistance genes Pm2, Pm19, Pm34, Pm35, PmY201, PmY212,
and PmM53 (McIntosh and Baker 1970, Lutz et al 1995, Miranda et al. 2006, Miranda et al
2007, Sun et al. 2006, Li et al. 2011).

Synthetic hexaploid wheat
Synthetic hexaploid wheat (SHW) is developed by crossing tetraploid wheat with Ae.
tauschii and followed by colchicine treatment or spontaneous meiotic restitution to obtain a full
complement of chromosomes (McFadden and Sears 1947, Figure 1.2A). Typically, T. turgidum
subsp. durum L. is used as the tetraploid parent because durum wheat is a domesticated, freethreshing wheat that is still widely grown. Alternatively, T. turgidum subsp. dicoccoides is used
as a wild, hulled tetraploid parent to simulate the natural hybridization that may have occurred
when hexaploid wheat was domesticated (Yang et al. 2009). In the last 30 years, the international
breeding group, CIMMYT has developed more than 1,000 SHW populations that have been used
extensively to improve wheat around the globe (Dreisigacker et al. 2008). One disadvantage of
using SHW is that the entire D genome of Ae. tauschii is transferred—a digression to an

12

Figure 1.2 Diagrams of A) synthetic hexaploid wheat development and B) direct
hybridization between Ae. tauschii and hexaploid wheat. Shades of purple, green, and blue
represent homoeologous A, B, and D genomes, respectively. In steps involving hybridization,
female parents are on the left and males are on the right. Encircled X = self-hybridization,
forward slash = recombination between homologous chromosomes. This figure was adapted
from Cox 1997 and Ogbonnaya et al. 2013.

13

unadapted D genome unsuitable for cultivation. Another disadvantage is that the A and B
genomes of the tetraploid wheat parent do not necessarily have good combinability with the D
genome of Ae. tauschii, whereas the A, B, and D genomes of hexaploid wheat have co-evolved
for thousands of years (Ogbonnaya et al. 2013). To address these limitations, primary SHW is
usually backcrossed to well-adapted hexaploid wheat before it is used for commercial wheat
breeding objectives (Figure 1.2A).

Direct hybridization
An alternative approach to SHW is the use of direct hybridization between Ae. tauschii
and hexaploid wheat followed by recurrent backcrossing to hexaploid wheat (Figure 1.2B).
Using this method, Gill and Raupp (1987) first demonstrated that F1 aneuploid embryos lacking
typical endosperm could be rescued on culture media and backcrossed with hexaploid wheat to
restore fertility. Then, by using BC1F1 individuals as pollen donors in a second backcross to
hexaploid wheat, it was possible to select against aneuploid gametes and restore euploid
chromosome segregation (Cox 1997). The primary advantage of this method is that stable,
recombinant D genome chromosomes can be recovered without perturbation of the
homoeologous A and B genomes of hexaploid wheat (Ogbonnaya et al. 2013). Additionally, if
the same inbred hexaploid wheat line is used for recurrent backcrossing, the genetic background
will be identical to that of the recurrent parent with the exception of introgressed D genome Ae.
tauschii loci (Figure 2B). In this way, the allohexaploid wheat genome is effectively reduced to
diploid segregation of D genome alleles, and the complex interactions between co-evolved
homoeologous genomes are retained.

14

Advancements in genetic and genomic tools for wheat
Genome sequencing
Sequencing the genomes of wheat and its wild relatives represents the most significant
advancements in the field of wheat breeding and genetics in recent years. Initially, due to the
immense size (17-gigabases) and complex structure of the allohexaploid wheat genome,
ditelosomic chromosome arms were isolated using flow-cytometry, sequenced, and assembled
individually to produce the first partial reference genome of wheat (The International Wheat
Genome Sequencing Consortium 2014). A year later, the first whole-genome shotgun assembly
of wheat was released by Chapman et al. (2015). This assembly was anchored to a dense genetic
map, but not annotated and only ~50% of the genome was represented. Finally, with longer reads
and improved assembly algorithms designed for large complex genomes, a new whole-genome
shotgun assembly of wheat was released that represents nearly 80% of the wheat genome
(Clavijo et al. 2017). Due to the smaller size of the diploid Ae. tauschii and T. urartu genomes,
whole-genome shotgun assemblies of each were released to the public in 2013 (Jia et al. 2013,
Ling et al. 2013). Also, a 4-gigabase physical map of Ae. tauschii was developed by Luo et al.
(2012) by finger printing bacterial artificial chromosomes and assembling/mapping contigs using
a 10K Illumina® Infinium SNP array. More recently, using long-read single-molecule
sequencing technology in conjunction with short reads, the Ae. tauschii genome was
independently re-sequenced and -assembled to improve contig length (Zimin et al. 2017).
Equipped with these genomic resources, substantial progress can now be made towards further
understanding the origins of wheat, the genetic diversity present in wheat and its wild relatives,
and the genetics underlying wheat response to abiotic and biotic stresses.

15

Wheat genotyping platforms
The primary genotyping tool used by wheat breeders and researchers in the early 2000’s
were microsatellite markers (namely simple sequence repeats). A gradual shift towards single
nucleotide polymorphism (SNP) genotyping platforms has since occurred. The first wheat
microsatellite map was produced in 1998 by Roder et. al. and was composed of 279
microsatellites. An improved wheat microsatellite map was released in 2004 with a total of 1,235
microsatellite loci mapped at an average interval distance of 2.2 cM (Somers et. al. 2004). At
that time, microsatellite markers were the most affordable marker technology, and highthroughput capillary electrophoresis allowed researchers to map these polymorphic co-dominant
markers in relatively large populations. Although microsatellite markers remain an important
technology for applications in marker assisted selection (MAS) and trait mapping, recent
advances in SNP genotyping have led to the large-scale adoption of SNP genotyping platforms
including genotyping-by-sequencing (GBS), SNP arrays, and KASP™ markers. The primary
advantage offered by these newer SNP genotyping platforms is higher throughput and increased
genome-wide marker coverage (Mammadov et al. 2012). Furthermore, decreased sequencing
cost, more user-friendly bioinformatics software, and increased genotyping platform flexibility
are making SNP marker data more accessible for all crop species, including wheat (Poland and
Rife 2012).

Genotyping-by-sequencing
The wheat breeding and research community has been very receptive to GBS technology
because it offers a cost-effective method to genotype many plants at several thousand loci.
Unlike other SNP genotyping platforms, no prior sequence data is needed for genotyping. This

16

allows GBS to be applied to novel germplasm and provides versatility. GBS was first
demonstrated by Elshire et. al. in 2011. The basic method for GBS involves DNA digestion by
restriction enzymes, adapter and barcode ligation, sample pooling and amplification, sequencing,
and SNP calling. One important advancement in GBS was the application in wheat by Poland et.
al. in 2012 using a novel two-enzyme approach. This approach included a rare-cutting restriction
enzyme and a common-cutting enzyme which together resulted in higher specificity and
reproducibility. While GBS has many advantages over alternative genotyping platforms, there
are many disadvantages too: missing data, non-uniform distribution of sequence data, sequencing
errors, SNP identification in duplicated loci, and the necessity for bioinformatics expertise when
analyzing GBS data (Beissinger et. al. 2013, Kim et al. 2016). Development of more accessible
GBS analysis software, SNP filtering, and a variety of data imputation methods have attempted
to mitigate these issues with mixed success (Rutkoski et. al. 2013, Glaubitz et. al. 2014, Limborg
et al. 2016).

KASP™ Markers
KASP™ markers are an important newer technology used to assay specific SNPs using a
competitive allele-specific polymerase chain reaction (Smith and Maughan 2015). Two forward
primers and a common reverse primer compete to amplify a specific segment of DNA. The two
forward primers are identical except for the 3’ nucleotide, which is the SNP that differentiates
one allele from the other. Two fluorescent dyes are included in the mix, with one binding to the
product from one allele and the other binding to the product from the alternative allele. Using a
fluorimeter, the signals from a given reaction can be measured. Individuals that express a single
fluorescence signal are homozygous for that allele, and individuals that express a combination of

17

both fluorescence signals are heterozygous. The substantial reduction in missing data points and
its use as a targeted approach to saturate regions of interest with markers are the primary
advantages of this technology (Rasheed et al. 2016). KASP™ markers were first used effectively
in wheat in 2011 when Allen et. al. identified 1,114 SNPs and designed KASP™ markers to
genotype 23 varieties. Many researchers have also found KASP™ markers to be complimentary
to GBS; while GBS is effective at identifying important polymorphisms, KASP™ markers can
be used to assay individuals with unknown genotypes (Gao et al. 2015, Lin et al. 2015). The
primary disadvantages of KASP™ markers are the need for prior SNP identification, higher cost
and lower throughput compared to other SNP platforms, and patents that limit the use of the
technology (Ertiro et al. 2015, www.lgcgroup.com/products/kasp-genotyping-chemistry).

Problem definition
As the demand for wheat increases and virulent plant pathogens threaten global wheat
production, breeders must accelerate genetic gain by increasing genetic diversity and rapidly
integrating novel resistance loci into elite varieties. While this is a challenging task, elegant
population development, improved genomic resources, and higher-throughput SNP genotyping
will facilitate ongoing efforts. By hybridizing Ae. tauschii directly with hexaploid wheat and
using the same elite wheat variety for recurrent backcrossing, forward breeding with Ae. tauschii
is possible. The effect of Ae. tauschii alleles can be characterized and mapped in hexaploid
wheat without the confounding effects of homoeologous chromosome segregation, the exact
genomic context can be determined using new reference genomes, and more accurate marker
assisted selection is enabled by contemporary SNP markers. Together, these advancements will

18

streamline the development of disease resistant wheat germplasm using foreign introgressions
from Ae. tauschii.

Objectives
To characterize and map genetic variation for stem rust and powdery mildew disease resistance
from Ae. tauschii in hexaploid wheat and to develop disease resistant germplasm and genetic
markers for ongoing breeding efforts.

19

REFERENCES

20

REFERENCES

Acevedo-Garcia J, Kusch S, Panstruga R (2014) Magical mystery tour: MLO proteins in plant
immunity and beyond. New Phytol. 204:273-281. doi: 10.1111/nph.12889
Acevedo-Garcia J, Spencer D, Thieron H, Reinstadler A, Hammond-Kosack K, Phillips A,
Panstruga R (2017) mlo-based powdery mildew resistance in hexaploid bread wheat
generated by a non-transgenic TILLING approach. Plant Biotechnol. J. 15:367-378. doi:
10.1111/pbi.12631
Allen AM, Barker G, Berry ST, Coghill JA, Gwilliam R, Kirby S, Robinson P, Brenchley RC,
D’Amore R, McKenzie N, Waite D, Hall A, Bevan M, Hall N, Edwards KJ (2011)
Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in
hexaploid bread wheat (Triticum aestivum L.). Plant Biotechnol. J. 9:1086-1099.
Alyor DE (2003) Spread of plant disease on a continental scale: role of aerial dispersal of
pathogens. Ecology 84(8):1989-1997.
Baidouri ME, Murat F, Veyssiere M, Molinier M, Flores R, Burlot L, Alaux M, Quesneville H,
Pont C, Salse J (2017) Reconciling the evolutionary origin of bread wheat (Triticum
aestivum). New Phytol. 213(3):1477-1486. doi: 10.1111/nph.14113
Beissinger TM, Hirsch CN, Sekhon RS, Foerster JM, Johnson JM, Muttoni G, Vaillancourt B,
Buell CR, Kaeppler SM, de Leon N (2013) Marker density and read depth for genotyping
populations using genotyping-by-sequencing. Genetics 193:1073-1081
Bhattacharya S (2017) Deadly new wheat disease threatens Europe’s crops. Nature 542:145-146.
doi: 10.1038/nature.2017.21424
Borner A, Ogbonnaya FC, Roder MS, Rasheed A, Periyannan S, Lagudah ES (2015) Aegilops
tauschii introgressions in wheat. In: Alien Introgression in Wheat. Springer International,
Switzerland, p. 245-271
Boutrot F, Zipfel C (2017) Function, discovery, and exploitation of plant pattern recognition
receptors for broad-specturm disease resistance. Annu. Rev. Phytopathol. 55:11.1-11.30.
doi: 10.1146/annurev-phyto-080614-120106
Brenchley R, Spannagl M, Pfeifer M, Barker GLA, D’Amore R, et al. (2012) Analysis of the
bread wheat genome using whole-genome shotgun sequencing. Nature 491:705-710. doi:
10.1038/nature11650
Brown JKM (2015) Durable resistance of crops to disease: a Darwinian perspective. Annu. Rev.
Phytopathol. 53:513-539. doi: 10.1146/annurev-phyto-102313-045914

21

Chapman J, Mascher M, Buluc A, Barry K, Georganas E, Session A, Strnadova V, Jenkins J,
Sehgal S, Oliker L, Schmutz J, Yelick KA, Scholz U, Waugh R, Poland JA, Muehlbauer
GJ, Stein N, Rokhsar DS (2015) A whole-genome shotgun approach for assembling and
anchoring the hexaploid bread wheat genome. Genome Biol. 16:26. doi: 10.1186/s13059015-0582-8
Clavijo BJ, Venturini L, et al. (2017) An improved assembly and annotation of the allohexaploid
wheat genome identifies complete families of agronomic genes and provides genomic
evidence for chromosomal translocations. Genome Res. 27:885-896. doi:
10.1101/gr.217117.116
Cowger C, Miranda L, Griffey C, Hall M, Murphy JP, Maxwell J (2012) Wheat powdery
mildew. In: Disease resistance in wheat. CAB International, Oxfordshire, p. 84-119
Cox TS (1997) Deepening the wheat gene pool. J. of Crop Prod. 1(1):1-25. doi:
10.1300/J144v01n01_01
Cui H, Tsuda K, Parker JE (2015) Effector-triggered immunity: from pathogen perception to
robust defense. Annu. Rev. Plant Biol. 66:487-511. doi: 10.1146/annurev-arplant050213-040012
Curtis R, Halford NG (2014) Food security: the challenge of increasing wheat yield and the
importance of not compromising food safety. Ann. Appl. Biol. 164:354-372. doi:
10.1111/aab.12108
Dimmock JPRE, Gooding MJ (2002) The influence of foliar diseases, and their control by
fungicides, on the protein concentration in wheat grain: a review. J. Agric. Sci. 138:349366. doi: 10.1017S0021859602002058
Dreisigacker S, Kishii M, Lage J, Warburton M (2008) Use of synthetic hexaploid wheat to
increase diversity for CIMMYT bread wheat improvement. Aust. J. Agric. Res. 59:413420. doi: 10.1071/AR07225
Dubcovsky J, Dvorak J (2007) Genome plasticity a key factor in the success of polyploid wheat
under domestication. Science 316(5833):1862-1866 doi: 10.1126/science.1143986
Dvorak J, Luo MC, Yang ZL and Zhang HB (1998) The structure of the Aegilops tauschii
genepool and the evolution of hexaploid wheat. Theor. Appl. Genet. 97: 657-670
Ellis JG, Lagudah ES, Spielmeyer W, Dodds PN (2014) The past, present and future of breeding
rust resistant wheat. Front. Plant Sci. 5:641. doi: 10.3389/fpls.2014.00641
Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A
robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. Plos
One 6:5

22

Ertiro BT, Ogugo V, Worku M, Das B, Olsen M, Labuschagne M, Semagn K (2015)
Comparison of kompetitive allele specific PCR (KASP™) and genotyping by sequencing
(GBS) for quality control analysis in maize. BMC genomics 16:980. doi:
10.1186/s12864-015-2180-2
Feldman M, Levy AA (2005) Allopolyploidy – a shaping force in the evolution of wheat
genomes. Cytogenet. Genome Res. 109:250-258. doi: 10.1159/000082407
Feuillet C, Langridge P, Waugh R (2007) Cereal breeding takes a walk on the wild side. Trends
Genet. 24(1):24-32. doi: 10.1016/j.tig.2007.11.001
Gao L, Kielsmeier-Cook J, Bajgain P, Zhang X, Chao S, Rouse MN, Anderson JA (2015)
Development of genotyping by sequencing (GBS)- and array- derived SNP markers for
stem rust resistance gen Sr42. Mol. Breed. 35:207. doi: 10.1007/s11032-015-0404-4
Gill BS, Raupp WJ (1987) Direct genetic transfers from Aegilops squarrosa L. to hexaploid
wheat. Crop. Sci. 27:445-450
Glaubitz JC, Casstevens TM, Lu F, Harriman J, Elshire RJ, Sun Q, and Buckler ES (2014)
TASSEL-GBS: a high capacity genotyping by sequencing analysis Pipeline. Plos One 9:2
Jia J, Zhao S, Kong X, Li Y, Zhao G, He W, Appels R et al. (2013) Aegilops tauschii draft
genome sequence reveals a gene repertoire for wheat adaptation. Nature 496:91-95. doi:
10.1038/nature12028
Jin Y, Szabo LJ, Pretorius ZA, Sing RP, Ward R, Fetch T (2008) Detection of virulence to
resistance gene Sr24 within race TTKS of Puccinia graminis f. sp. tritici Plant Dis.
92:923-926. doi: 10.1094/ PDIS-92-6-0923
Jin Y, Szabo LJ, Rouse MN, Fetch T, Pretorius ZA, Wanyera R, Njau P (2009) Detection of
virulent to resistance gene Sr36 within the TTKS race lineage of Puccinia graminis f. sp.
tritici. Plant Dis. 93:367-370. doi: 10.1094/ PDIS-93-4-0367
Jin Y (2011) Role of Berberis spp. as alternate hosts in generating new races of Puccinia
graminis and P. striiformis. Euphytica 179:105-108. doi: 10.1007/s10681-010-0328-3
Kihara H (1944) Discovery of the DD-analyser, one of the ancestors of Triticum vulgare (in
Japanese). Agric. Hortic. 19:1742-1749
Kim C, Guo H, Kong W, Chandnani R, Shuang L, Paterson AH (2016) Application of
genotyping by sequencing technology to a variety of crop breeding programs. Plant Sci.
242:14-22. doi: 10.1016/j.plantsci.2015.04.016
Krattinger SG, Lagudah ES, Spielmeyer W, Sing RP, Huerta-Espino J, McFadden H, Bossolini
E, Selter LL, Keller B (2009) A putative ABC transporter confers durable resistance to

23

multiple fungal pathogens in wheat. Science 323(5919):1360-1363. doi:
10.1126/science.ll66453
Limborg MT, Seeb LW, Seeb JE (2016) Sorting duplicated loci disentangles complexities of
polyploid genomes masked by genotyping by sequencing. Mol. Ecol. 25:2117-2129.
Ling H, ZhaoS, Liu D, Wang J, Sun H, Zhang C (2013) Draft genome of the wheat A-genome
progenitor Triticum urartu. Nature 496:87-90. doi: 10.1038/nature11997
Lin M, Cai S, Wang S, Liu S, Zhang G, Bai B (2015) Genotyping-by-sequencing (GBS)
identified SNP tightly linked to QTL for pre-harvest sprouting resistance. Theor. Appl.
Genet. 128:1385-1395. doi: 10.1007/s00122-015-2513-1
Li X, Kapos P, Zhang Y (2015) NLRs in plants. Curr. Opin. Immunol. 32:114-121. doi:
10.1016/j.coi.2015.01.014
Li Z, Lan C, He Z, Singh RP, Rosewarne GM, Chen X, Xia X (2014) Overview and application
of QTL for adult plant resistance to leaf rust and powdery mildew in wheat. Crop Sci.
54:1907-1925. doi: 10.2135/cropsci2014.02.0162
Li T, Zhang Z, Hu Y, Duan X, Xin Z (2011) Identification and molecular mapping of a
resistance gene to powdery mildew from the synthetic wheat line M53. J. Appl. Genet.
52:137–143. doi: 10.1007/s13353-010-0006-0
Luo MC, Gu YQ, You FM, Deal KR, Ma Y, Hu Y, Juo N, Wang Y, Wang J, Chen S, Jorgensen
CM, Zhang Y, McGuire PE, Pasternak S, Stein JC, Ware D, Kramer M, McCombie WR,
Kianian SF, Martis MM, Mayer KFX, Sehgal SK, Li W, Gill B, Bevan MW, Simkova H,
Dolezel J, Weining S, Lazo GR, Anderson OD, Dvorak J (2013) A 4-gigabase physical
map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the
wheat D-genome progenitor. PNAS 110(19):7940-7945.
Lutz J, Hsam SLK, Limpert E, Zeller FJ (1995) Chromosomal location of powdery mildew
resistance genes in Triticum aestivum L. (common wheat). 2. Genes Pm2 and Pm19 from
Aegilops squarrosa L. Heredity (Edinb) 74:152–156. doi: 10.1038/hdy.1995.22
Mago R, Zhang P, Vautrin S, Simkova H, Bansal U, Luo M, Rouse M, Karaoglu H, Periyannan
S, Kolmer J, Jin Y, Ayliffe MA, Bariana H, Park RF, McIntosh R, Doezel J, Berges H,
Spielmeyer W, Lagudah ES, Ellis JG, Dodds PN (2015) The wheat Sr50 gene reveals
rich diversity at a cereal disease resistance locus. Nat. Plants 1:15186. doi:
10.1038/NPLANTS.2015.186
Mammadov J, Aggarwal R, Buyyarapu R, Kumpatla (2012) SNP markers and their impact on
plant breeding. Int. J. Plant Genomics. doi: 10.1155/2012/728398
McFadden ES, Sears ER (1946) The origin of Triticum spelta and its free-threshing hexaploid
relatives. J. Hered. 37(3): 81-89.
24

McFadden ES, Sears ER (1947) The genome approach in radical wheat breeding. J. Am. Soc.
Agron. 39:1011-1026.
Mcintosh RA, Baker EP (1970) Cytogenetical studies in wheat IV. chromosome location and
linkage studies involving the Pm2 locus for powdery mildew resistance. Euphytica
19:71–77.
McIntosh RA, Wellings CR, Park RF (1995) Wheat rusts: an atlas of resistance genes. CSIRO
Publications, East Melbourne, p. 1-6
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. doi:
10.1007/s00122-007-0530-4
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. doi: 10.1007/s00122-006-0397-9
Mizuno N, Yamasaki M, Matsuoka Y, Kawahara T and Takumi S (2010) Population structure of
wild wheat D-genome progenitor Aegilops tauschii Coss.: implications for intraspecific
lineage diversification and evolution of common wheat. Mol. Ecol. 19:999-1013. doi:
10.1111/j.1365-294X.2010.04537.x
Nejat N, Rookes J, Mantri N, Cahill DM (2016) Plant-pathogen interactions: toward
development of next-generation disease-resistant plants. Crit. Rev. Biotechnol.
37(2):229-237. doi: 10.3109/07388551.2015.1134437
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.
Ogbonnaya FC, Abdalla O, Mujeeb-Kazi A, Kazi AG, Xu SS, Gosman N, Lagudah ES, Bonnett
DG, Sorells ME, Tsujimoto H (2013) Synthetic hexapliods: harnessing species of
primary gene pool for wheat improvement. Plant Breed. Rev. 37:35-122
Olson EL, Rouse MN, Pumphrey MO, Bowden RL, Gill BS, Poland JA (2013a) Simultaneous
transfer, introgression, and genomic localization of genes for resistance to stem rust race
TTKSK (Ug99) from Aegilops tauschii to wheat. Theor. Appl. Genet. 126:1179-1188.
doi: 10.1007/s00122-013-2045-5
Olson EL, Rouse MN, Pumphrey MO, Bowden RL, Gill BS, Poland JA (2013b) Introgression of
stem rust resistance genes SrTA10187 and SrTA10171 from Aegilops tauschii to wheat.
Theor. Appl. Genet. 126:2477-2484. doi: 10.1007/s00122-013-2148-z

25

Panstruga R (2003) Establishing compatibility between plants and obligate biotrophic pathogens.
Curr. Opin. Plant Biol. 6:320-326. doi: 10.1016/S1369-5266(03)00043-8
Parks R, Carbone I, Murphy JP, Marshall D, Cowger (2008) Virulence structure of the eastern
U.S. wheat powdery mildew population. Plant Dis. 92:1074-1082. doi: 10.1094/ PDIS92-7-1074
Periyannan S, Bansal U, Bariana H, Deal K, Luo M, Dvorak J, Lagudah E (2014) Identification
of a robust molecular marker for the detection of the stem rust resistance gene Sr45 in
common wheat. Theor. Appl. Genet. 127:947-955. doi: 10.1007/s00122-014-2270-6
Periyannan S, Moore J, Ayliffe M, Bansal U, Wang X, Huang L, Deal K, Luo M, Kong X,
Bariana H, Mago R, McIntosh R, Dodds P, Dvorak J, Lagudah E (2013) The gene Sr33,
an ortholog of barley Mla genes, encodes resistance to wheat stem rust race Ug99.
Science 341(6147):786-788. doi: 10.1126/science.1239028
Pestsova E, Korzun V, Goncharov NP, Hammer K, Ganal MW, Roder MS (2000) Microsatellite
analysis of Aegilops tauschii germplasm. Theor. Appl. Genet. 101:100-106.
Petersen RH (1974) The rust fungus life cycle. Bot. Rev. 40(4):453-513.
Peterson PD, Leonard KJ, Miller JD, Laudon RJ, Sutton TB (2005) Prevalence and distribution
of common barberry, the alternate host of Puccinia graminis, in Minnesota. Plant Dis.
89:159-163. doi: 10.1094/PD-89-0159
Petit-Houdenot Y, Fudal I (2017) Complex interactions between fungal avirulence genes and
their corresponding plant resistance genes and consequences for disease resistance
management. Front. Plant Sci. 8:1072. doi: 10.3389/fpls.2017.01072
Poland JA, Balint-Kurti PJ, Wisser RJ, Pratt RC, Nelson RJ (2008) Shades of gray: the world of
quantitative disease resistance. Trends Plant Sci. 14(1):21-29. doi:
10.1016/j.tplants.2008.10.006
Poland JA, Brown PJ, Sorrells ME, and Jannink J (2012) Development of high-density genetic
maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing
approach. Plos One 7:2
Poland JA, Rife TW (2012) Genotyping-by-sequencing for plant breeding and genetics. Plant
Genome 5(2):92- 102
Rasheed A, Wen W, Gao F, Zhai S, Jin H, Liu J, Guo Q, Zhang Y, Dreisigacker S, Xia X, He Z
(2016) Development and validation of KASP™ assays for genes underpinning key
economic traits in bread wheat. Theor. Appl. Genet. 129:1843-1860. doi:
10.1007/s00122-016-2743-x

26

Reif JC, Zhang P, Dreisigacker S, Warburton ML, van Ginkel M, Hoisington D, Bohn M,
Melchinger AE (2005) Wheat genetic diversity trends during domestication and breeding.
Theor. Appl. Genet. 110:859-864
Roder MS, Korzun V, Wandehake K, Planschke J, Tixier MH, Leroy P, Ganal MW (1998) A
microsatellite map of wheat. Genetics 149:2007-2023
Rouse MN, Olson EL, Gill BS, Pumphrey MO, Jin Y (2011) Stem rust resistance in Aegilops
tauschii germplasm. Crop Sci. 51:2074-2078. doi: 10.2135/cropsci2010.12.0719
Rutkoski JE, Poland J, Jannink J, and Sorrells ME (2013) Imputation of unordered markers and
the impact on genomic selection accuracy. Genes Genomes Genet. 3:427-439
Saintenac C, Zhang W, Salcedo A, Rouse MN, Trick HN, Akhunov E, Dubcovsky J (2013)
Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group.
Science 341(6147):783-786. doi: 10.1126/science.1239022
Sanchez-Martin J, Steuernagel B, Ghosh S, Herren G, Hurni S, Adamski N, Vrana J, Kubalakova
M, Krattinger SG, Wicker T, Dolezel J, Keller B, Wulff BH (2016) Rapid gene isolation
in barley and wheat by mutant chromosome sequencing. Genome Biol. 17:221. doi:
10.1186/s13059-016-1082-1
Schulze-Lefert P, Vogel J (2000) Closing the ranks to attack by powdery mildew. Trends Plant
Sci. Rev. 5(8):343-348
Schumann, G.L., C.J. D’Arcy (2012) Essential Plant Pathology. Am. Phytopathol. Soc., St. Paul,
MN.
Shewry P.R. (2009) Wheat. J. Exp. Bot. 60(6):1537-1553. doi: 10.1093/jxb/erp058
Singh RP, Hodson DP, Huerta-Espino J, Jin Y, Bhavani S, Njau P, Herrera-Foessel S, Singh PK,
Singh S, Govindan V (2011) The emergence of Ug99 races of the stem rust fungus is a
threat to world wheat production. Annu. Rev. Phytopathol. 49:465-481. doi:
10.1146/annurev-phyto-072910-095423
Smith SM, Maughan PJ (2015) SNP genotyping using KASPar assays. In: Plant genotyping:
methods and protocols. Springer Science, New York, p. 243-256
Somers DJ, Isaac P, and Edwards K (2004) A high-density microsatellite consensus map for
bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 109:1105-1114
Steuernagel B, Periyannan SK, Hernandez-Pinzon I, Witek K, Rouse MN, Yu G, Hatta A,
Ayliffe M, Bariana H, Jones JDG, Lagudah ES, Wulff BBH (2016) Rapid cloning of
disease-resistance genes in plants using mutagenesis and sequence capture. Nat.
Biotechnol. 34(6):652-655. doi: 10.1038/nbt.3543

27

Sun X-L, Liu D, Zhang H-Q, Huo N-X, Zhou R-H, Jia J-Z (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. doi: 10.1111/j.1672-9072.2006.00328.x
Te Beest DE, Paveley ND, Shaw MW, van den Bosch F (2008) Disease-weather relationships
for powdery mildew and yellow rust on winter wheat. Phytopathology 98:609-617. doi:
10.1094/ PHYTO-98-5-0609
The International Wheat Genome Sequencing Consortium (2014) A chromosome-based draft
sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 345(6194).
doi: 10.1126/science.1251788
Wang JW, Luo M, Chen Z, You FM, Wei Y, Zheng Y, and Dvorak J (2013) Aegilops tauschii
single nucleotide polymorphisms shed light on the origins of wheat D-genome genetic
diversity and pinpoint the geographic origin of hexaploid wheat. New Phytol. 198:925937
Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qui J (2014) Simultaneous editing of three
homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.
Nat. Biotechnol. 32(9):947-951. doi: 10.1038/nbt.2969
Yahiaoui N, Srichumpa P, dudler R, Keller B (2004) Genome analysis at different ploidy levels
allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat. Plant
J. 37:528-538. doi: 10.1046/j.1365-313X.2003.01977.x
Yang W, Liu D, Li J, Zhang L, Wei H, Hu X, Zheng Y, He Z, Zou Y (2009) Synthetic hexaploid
wheat and its utilization for wheat genetic improvement in China. J Genet. Genomics
36:539-546. doi: 10.1016/S1673-8527(08)60145-9
Yu G, Zhang Q, Friesen TL, Rouse MN, Jin Y, Zhong S, Rasmussen JB, Lagudah ES, Xu SS
(2015) Identification and mapping of Sr46 from Aegilops tauschii accession CIae 25
conferring resistance to race TTKSK (Ug99) of wheat stem rust pathogen. Theor. Appl.
Genet. 128:431-443. doi: 10.1007/s00122-014-2442-4

28

CHAPTER II
Fine mapping of the stem rust resistance gene SrTA10187

The Ug99-effective stem rust resistance gene SrTA10187 was fine-mapped to a 1.1 cM interval
on 6DS, candidate disease resistance genes were identified, and molecular markers were
developed for ongoing wheat breeding efforts. For a full text of this work go to: Theoretical and
Applied Genetics, 2016, 129(12):2369-2378, doi: 10.1007/s00122-016-2776-1

29

CHAPTER III
Identification of Pm58 from Aegilops tauschii

Using a population of wheat-Aegilops tauschii introgression lines, the novel powdery mildew
resistance gene Pm58 was mapped to chromosome 2DS and confirmed to be effectie under field
conditions. Additionally, molecular markers were developed for ongoing wheat breeding efforts.
For a full text of this work go to: Theoretical and Applied Genetics, 2017, 130:1123-1133, doi:
10.1007/s00122-017-2874-8

30

CHAPTER IV
Registration of Two Wheat Germplasm Lines Fixed for Pm58

Journal of Plant Registrations, June 2017, under review
Andrew T. Wiersma1, Rebecca B. Whetten2, Guorong Zhang3, Sunish K. Sehgal4, Frederic L.
Kolb5, Jesse A. Poland6, R. Esten Mason7, Arron H. Carter8, Christina Cowger2,9, Eric L.
Olson1*

1

Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI

2

USDA-ARS Plant Science Research, Raleigh, NC

3

Agriculture Research Center, Kansas State University, Hays, KS

4

Department of Agronomy, Horticulture, and Plant Science, South Dakota State University,

Brookings, SD
5

Department of Crop Sciences, University of Illinois, Champaign, IL

6

Department of Agronomy, Kansas State University, Manhattan, KS

7

Department of Crop, Soil and Environmental Science, University of Arkansas, Fayetteville, AR

8

Department of Crop and Soil Sciences, Washington State University, Pullman, WA

9

Department of Plant Pathology, North Carolina State University

31

Abstract
Powdery mildew, caused by Blumeria graminis (D.C.) f. sp. tritici, is a persistent threat to global
wheat (Triticum aestivum L.) production. To broaden the genetic base for resistance to powdery
mildew in wheat, germplasm lines U6714-A-011 and U6714-B-056 were developed at Michigan
State University and are fixed for the novel powdery mildew resistance gene Pm58. This gene
was identified in Aegilops tauschii Coss. accession TA1662, introgressed, and mapped to wheat
chromosome 2DS. The two germplasm lines described are BC2F4-derived inbred backcrossed
lines from a direct cross between TA1662 and the recurrent wheat parent KS05HW14, a hard
white winter wheat line adapted to western Kansas. In addition to exhibiting resistant reactions to
multiple Bgt isolates with broad virulence profiles, both lines have moderate yield potential and
good agronomic characteristics, making them suitable as breeding germplasm. The availability
of these lines will enable the incorporation of Pm58 into wheat breeding programs, providing
additional genetic variation for resistance to powdery mildew.

Introduction
As the causal agent of powdery mildew, the biotrophic ascomycete Blumeria graminis f.
sp. tritici (Bgt) remains among the most important fungal pathogens affecting wheat (Triticum
aestivum L.). The potential for large-scale grain yield loss due to powdery mildew and the
breakdown of host resistance necessitate ongoing discovery of additional sources of genetic
resistance to Bgt (Cowger et al. 2012).
The wild wheat relative Aegilops tauschii Coss. is an important source of genes
conferring resistance to biotrophic pathogens, including powdery mildew. Due to D genome
homology between Ae. tauschii and T. aestivum, chromosomes from interspecific crosses

32

recombine normally, allowing for migration of alleles by meiotic recombination. One of the most
widely deployed powdery mildew resistance genes in wheat, Pm2, was derived from Ae. tauschii
(McIntosh and Baker 1970, Bennett 1984), and multiple Ae. tauschii accessions have been used
as powdery mildew resistance donors to improve germplasm (Murphy et al. 1998, Murphy et al.
1999).
In the ongoing effort to improve powdery mildew resistance in wheat, Wiersma et al.
(2017) identified and mapped the novel powdery mildew resistance gene Pm58. The resistance
donor, Ae. tauschii accession TA1662 from Azerbaijan, was crossed directly with the susceptible
hard white wheat line KS05HW14 and lines fixed for Pm58 were identified. The objectives of
this study are to characterize powdery mildew resistance and grain yield in two germplasm lines,
U6714-A-011 and U6714-B-056, fixed for Pm58. Availability of these well-adapted germplasm
lines to the breeding community will facilitate the broadening of the genetic base for powdery
mildew resistance in wheat.

Methods
Germplasm line development
The susceptible wheat line KS05HW14 (KS98HW452/CO960293//KS920709B-5-2),
was used as the wheat parent in a direct interspecific cross with the powdery mildew-resistant
Ae. tauschii accession TA1662 (Gill and Raupp 1987, Wiersma et al. 2017). The hard white
winter wheat line KS05HW14 was developed by Dr. Joe Martin of Kansas State University in
Hays, KS. The powdery mildew resistance donor, TA1662 is an Ae. tauschii accession collected
from Azerbaijan and maintained by the Wheat Genetics Resource Center at Kansas State
University. Following embryo rescue, F1 hybrids were backcrossed to KS05HW14 as females,

33

and a single BC1F1 plant with powdery mildew resistance was backcrossed again to KS05HW14.
A population of BC2F1-derived plants were maintained by single-seed-descent to the BC2F4
generation when seed of BC2F4-derived head rows was produced in 2014. Yield trials and
powdery mildew tests were conducted in subsequent generations. The two genotypes U6714-A011 and U6714-B-056 were identified as being fixed for Pm58 based on powdery mildew
disease scores from field studies and detached-leaf assays (Wiersma et al. 2017). U6714-A-011
and U6714-B-056 are also homozygous for the TA1662 haplotype at the KASP™ marker loci KTP127986 to K-TP69304.

Powdery mildew evaluation
Isolate-specific reactions of U6714-A-011, U6714-B-056, KS05HW14, and the
susceptible check cultivar Jagalene to 20 Bgt isolates were tested using detached-leaf assays by
the USDA-ARS Plant Science Research Unit in Raleigh, North Carolina. The 20 Bgt isolates
were selected based on broad geographic distribution and virulence profiles (Cowger et al.
2017). Detached leaf segments (10-12 days, 1.5 cm long) were floated on 0.5% (w/v) water agar
amended with benzimidazole (50 mg L-1) (Parks et al. 2008). Two replicate leaf segments of
U6714-A-011, U6714-B-056, and KS05HW14, and four replicate leaf segments of Jagalene
were rated on each plate. Each plate was inoculated with an individual Bgt isolate, and four
plates total were rated for each isolate. Leaf segments were rated 10 to 11 days post inoculation
using a 0-9 rating system described previously by Parks et al. 2008, where ratings 0-3, 4-6, and
7-9 are classified as resistant, intermediate, or susceptible, respectively.

34

Agronomic evaluation
Yield trials were grown in three locations in 2015: Hays, KS; Ashland, KS; and
Richville, MI. In 2016, four additional yield trial locations were included: Brookings, SD;
Champaign, IL; Marianna, AR; and Pullman, WA. Yield trial fertilization varied by location and
fungicides were applied at several locations to control infection by stripe rust (Puccinia
striiformis f.sp. tritici). U6714-A-011 and U6714-B-056 were each planted in single replicate
plots in an augmented design containing six incomplete blocks. Replicated plots of KS05HW14,
and a locally adapted check were included in each block to control for variation. LS-means of
grain yield and 95% confidence intervals were calculated using a mixed linear model in
RStudio® (RStudio, Boston, MA, USA) using R version 3.2.1 and the packages lme4 (v.1.1-12)
and lsmeans (v.2.25-5). Genotype was treated as a fixed effect and block was treated as a random
effect.

Characteristics
Powdery mildew resistance
U6714-A-011 and U6714-B-056 were tested for their reaction to 20 Bgt isolates collected
from widely separated locations in hard and soft wheat growing regions of the central and
eastern United States (Table 4.1). Based on means of replicate leaf segments, U6714-A-011
expressed resistant to intermediate reactions to 15 Bgt isolates and susceptible reactions to 5
isolates; U6714-B-056 expressed resistant to intermediate reactions to 12 Bgt isolates and
susceptible reactions to 8 isolates. U6714-A-011 and U6714-B-056 had consistently lower scores
than both the recurrent wheat parent, KS05HW14, and the susceptible check, Jagalene (Table
4.1). In general, U6714-A-011 and U6714-B-056 were more resistant to powdery mildew

35

Table 4.1 Isolate-specific powdery mildew reactions identified in wheat lines U6714-A-011, U6714-B-056, KS05HW14, and the
Bgt susceptible check Jagalene. Powdery mildew disease severity was rated on a 0-9 scale, and the mean score of replicate leaf
segments is reported with the standard deviation in parenthesis.
Bgt isolate

Origin of isolate
(City, State, Year)

GAP-A-2-3
GAP-B-2-2
MIR(14)-D-3-3
MIR(14)-E-1-3
MSG-A-3-1
MSG-C-3-4
MTG1-1a
MTG1-3a
NCC-B-1-3
NCF-D-1-1
NEI-1-3
NEI-3-1
NEI-5-5
NYA-E-3-3
NYB-E-1-2
OKH-A-2-3
OKS-A-2-2
OKS-B-2-2
PAF(14)-D-1-2
PAF-E-2-2

Plains, GA, 2013
Plains, GA, 2013
Rogers City, MI, 2014
Rogers City, MI, 2014
Greenwood, MS, 2013
Greenwood, MS, 2013
Geraldine, MT, 2016
Geraldine, MT, 2016
Chocowinity, NC, 2013
Four Oaks, NC, 2013
Ithaca, NE, 2016
Ithaca, NE, 2016
Ithaca, NE, 2016
Aurora, NY, 2013
Brockport, NY, 2013
Hinton, OK, 2013
Stillwater, OK, 2013
Stillwater, OK, 2013
Pennsylvania Furnace, PA, 2014
Pennsylvania Furnace, PA, 2013

Avirulent/virulent on Pm genes†
Pm1a,1b,4b,16,17,36 / 2,3a,3b,4a,6,8
Pm1a,1b,4b,16,17,36 / 2,3a,3b,4a,6,8
Pm1a,1b,3b,4a,4b,17,34,37 / 2,3a,6,8,25,35
Pm1a,1b,2,3b,4a,4b,17,25,34,35,37 / 3a,6,8
Pm1a,1b,2,4a,4b,8,16,17,36 / 3a,3b,6
Pm1a,1b,4b,16,17,36 / 2,3a,3b,4a,6,8
Pm1a,1b,2,3a,3b,4a,4b,6,8,17,25,34,37 / 35
Pm1a,1b,3a,3b,4b,8,17,25,34,35,37 / 2,4a,6
Pm1a,1b,2,4b,8,16,36 / 3a,3b,4a,6,17
Pm1a,1b,2,4a,4b,16,17,36 / 3a,3b,6,8
Pm1a,1b,2,3a,4a,4b,25,34,35,37 / 3b,6,8,17
Pm1a,1b,2,3a,3b,4a,4b,17,25,34,35,37 / 6,8
Pm1a,1b,3a,3b,4b,8,25,34,37 / 2,4a,6,17,35
Pm1a,1b,4b,6,8,16,17,36 / 2,3a,3b,4a
Pm1a,1b,2,4b,16,17,36 / 3a,3b,4a,6,8
Pm1a,1b,2,3a,3b,4a,4b,8,16,17,36 / 6
Pm1a,1b,2,3a,3b,4b,16,36 / 4a,6,8,17
Pm1a,1b,2,3a,3b,4a,4b,16,17,36 / 6,8
Pm1a,1b,3b,4b,17,25,37 / 2,3a,4a,6,8,34,35
Pm1a,1b,4a,4b,8,16,17,36 / 2,3a,3b,6

U6714-A-011 U6714-B-056
(n = 8)
(n = 8)
6.4 (2.1)
6.1 (2.1)
7.5 (0.5)
7.9 (0.4)
7.5 (0.8)
5.1 (2.5)
4.4 (3.7)
3.0 (3.4)
4.1 (3.2)
8.0 (0.0)
3.8 (4.1)
3.1 (2.6)
6.4 (2.7)
6.9 (1.6)
6.8 (0.9)
3.1 (2.4)
2.4 (2.6)
5.8 (1.5)
7.9 (0.4)
4.3 (3.7)

7.5 (0.8)
7.5 (0.5)
7.4 (0.7)
7.4 (0.7)
7.0 (0.8)
5.0 (3.7)
5.1 (2.7)
5.5 (2.6)
5.3 (2.4)
7.8 (0.5)
4.3 (3.1)
6.3 (1.8)
5.4 (2.5)
6.8 (2.1)
7.1 (1.0)
1.9 (2.7)
3.0 (3.4)
6.1 (2.8)
8.0 (0.0)
5.9 (2.0)

KS05HW14
(n = 8)

Jagalene
(n = 16)

8.0 (0.0)
7.9 (0.4)
7.9 (0.4)
8.0 (0.0)
8.0 (0.0)
7.6 (0.7)
7.0 (2.8)
8.0 (0.0)
8.0 (0.0)
8.0 (0.0)
7.0 (2.8)
8.0 (0.0)
8.0 (0.0)
8.0 (0.0)
7.4 (0.7)
7.9 (0.4)
8.0 (0.0)
6.9 (2.8)
8.0 (0.0)
8.0 (0.0)

8.0 (0.0)
7.8 (0.4)
7.9 (0.3)
7.9 (0.3)
8.0 (0.0)
7.6 (0.8)
7.6 (0.6)
7.9 (0.3)
7.8 (0.4)
8.0 (0.0)
8.0 (0.0)
8.0 (0.0)
8.0 (0.0)
7.6 (0,5)
7.8 (0.4)
7.6 (0.5)
7.6 (0.5)
7.8 (0.4)
7.9 (0.3)
7.9 (0.3)

†Bgt isolate was considered avirulent if the single-gene differential line expressed a reaction <7.0. Genotypes with isolate-specific reactions less than 7 are
indicated in bold. n indicates the number of leaf segments rated.

36

isolates collected from the central states (Oklahoma, Nebraska, and Montana), compared to those
collected from eastern states. Higher standard deviations in disease scores were observed in
resistant and intermediate reactions compared to susceptible reactions (Table 4.1).

Agronomic evaluations
U6714-A-011 and U6714-B-056 are both free-threshing hard white winter wheat lines
that exhibit good agronomic characteristics. In Michigan during the 2015 and 2016 growing
seasons, the average plant height of both lines was 84 cm and anthesis occurred within one day
of the recurrent parent KS05HW14 (late May to early June). Plant type and early maturity were
stable and uniform across years and locations, indicating seed purity with very low levels of off
types or heterozygosity.
When yield was tested under multiple environments including locations in Kansas, South
Dakota, Illinois, Arkansas, Washington, and Michigan, U6714-A-011 and U6714-B-056 had
moderate grain yield potential (Table 4.2). With the exception of Hays, KS in 2015, the two
germplasm lines did not yield higher than the locally adapted check variety. On average, U6714B-056 had higher yield potential than U6714-A-011, but both lines yielded lower than the
recurrent parent KS05HW14 in all locations except Pullman, WA. In Pullman, WA, U6714-A011 yielded within the 95% confidence interval of KS05HW14, and U6714-B-056 had grain
yield higher than KS05HW14 (Table 4.2).

Discussion
The germplasm lines described here are the first publicly available wheat lines fixed for
the novel powdery mildew resistance gene Pm58 derived from Ae. tauschii. Based on its

37

Table 4.2 Grain yield LS-means of locally adapted check varieties, KS05HW14, U6714-A-011, and U6714-B-056 in 10 diverse environments
throughout the United States.
-----------------------------------------------------t ha-1----------------------------------------------------_____Locally adapted check varieties_____
_____KS05HW14_____
U6714-A-011
U6714-B-056
Location

Year

Name

Grain yield

95% CI

Grain yield

95% CI

Ashland, KS
Ashland, KS
Brookings, SD
Champaign, IL
Hays, KS
Hays, KS
Marianna, AR
Pullman, WA
Richville, MI
Richville, MI

2015
2016
2016
2016
2015
2016
2016
2016
2015
2016

Everest
Everest
Lyman
IL07-19334
Ernie
Joe
AR11LE24
Jasper
AC Mountain
Ambassador

4.64
4.85
3.60
7.85
2.73
6.76
4.12
9.06
5.18
5.63

4.45 - 4.82
4.65 – 5.05
3.30 - 3.90
7.47 - 8.22
2.34 - 3.12
6.65 - 6.87
3.86 - 4.39
8.75 - 9.37
5.09 - 5.26
5.31 - 5.95

4.66
4.20
3.30
5.67
3.22
3.61
2.73
4.45
4.95
5.31

4.41 - 4.89
4.11 – 4.30
3.17 - 3.44
5.50 - 5.85
3.05 - 3.40
3.56 - 3.66
2.60 - 2.85
4.31 - 4.59
4.76 - 5.15
5.15 - 5.46

Grain yield
(% KS05HW14)
3.54 (76%)
3.06 (73%)
0.36† (11%)
3.28 (58%)
1.51 (47%)
2.88 (80%)
2.52 (92%)
4.43 (100%)
4.32 (87%)
2.40 (45%)

Grain yield
(% KS05HW14)
3.23 (69%)
3.84 (91%)
1.64 (50%)
4.96 (87%)
3.36 (104%)
2.85 (79%)
2.27 (83%)
5.34‡ (120%)
4.12 (83%)
4.32 (81%)

†Exceptionally low yield was the result of a severe stripe rust and some difficulty threshing in Brookings, SD in 2016. ‡ Grain yield is significantly higher
than the recurrent parent, KS05HW14. CI = confidence interval.

38

performance against the present, geographically representative set of Bgt isolates, Pm58 is likely
to be particularly effective in the hard wheat region (Kansas, Nebraska, Oklahoma), where the
Bgt population has lower virulence complexity than in soft wheat growing areas (Cowger et al,
2017). However, our results indicate Pm58 also confers partial resistance to some isolates from
predominantly soft wheat-growing areas, and thus can be useful in those regions in combination
with other sources of resistance.
U6714-A-011 and U6714-B-056 have acceptable agronomic adaptation, perform well
under conventional wheat management, and can be used in the development of elite wheat lines
with improved powdery mildew resistance. Additionally, by using KASP™ markers linked to
Pm58 (Wiersma et al. 2017), plants can be selected in the absence of disease pressure and
multiple powdery mildew resistance genes can be pyramided simultaneously.

Availability
Small quantities of unrestricted seed are available immediately for distribution. Written
requests should be submitted to Dr. Eric L. Olson at Michigan State University, East Lansing,
MI. Breeder seed was produced in individual yield trial plots at the Michigan State University,
Saginaw Valley Research and Extension Center in Frankenmuth, MI (43.395, -83.676). Five
years from the publication date, the National Small Grains Collection (USDA-ARS) in
Aberdeen, Idaho is responsible for continued organization and maintenance of seed stocks. Seed
was also deposited in the USDA-ARS National Center for Genetic Resources Preservation
(NCGRP) in Ft. Collins, Colorado. It is requested that appropriate recognition be made if this
germplasm contributes to the development of a new breeding line or cultivar.

39

Acknowledgments
We would like to thank the Michigan Wheat Program for ongoing support of wheat breeding and
genetics research at Michigan State University. We would also like to thank our collaborators
and technical staff for phenotyping and yield testing these lines.

40

REFERENCES

41

REFERENCES

Bennett FGA (1984) Resistance to powdery mildew in wheat: a review of its use in agriculture
and breeding programs. Plant Pathol. 33:279-300
Cowger C, Miranda L, Griffey C, Hall M, Murphy JP, Maxwell J (2012) Wheat powdery
mildew. In: Disease resistance in wheat. CAB International, Oxfordshire, p. 84-119.
Cowger C, Mehra L, Arellano C, Meyers E, Murphy JP (2017) Virulence differences
in Blumeria graminis f. sp. tritici from the central and eastern United
States. Phytopathology: in review
Gill BS, Raupp WJ (1987) Direct genetic transfers from Aegilops squarrosa L. to hexaploid
wheat. Crop Sci. 27:445-450
Mcintosh RA, Baker EP (1970) Cytogenetical studies in wheat IV. Chromosome location and
linkage studies involving the Pm2 locus or powdery mildew resistance. Euphytica 19:7177
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 (1999) Registration of NC97BGTD7 and
NC97BGTD8 wheat germplasms resistant to powdery mildew. Crop Sci 39:884–885
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. doi: 10.1094/ PDIS92-7-1074
Wiersma AT, Pulman JA, Brown LK, Cowger C, Olson EL (2017) Identification of Pm58 from
Aegilops tauschii. Theor. Appl. Genet. doi: 10.1007/s00122-017-2874-8

42

Chapter V
Conclusions and future perspectives

43

Overview of dissertation research
High resolution mapping of the Ug99-effective stem rust resistance gene SrTA10187 was
accomplished using more than one thousand BC3F2 individuals and a combination of SNP, SSR,
and STS markers. A total of fifteen KAPS™ markers were designed based on SNPs available in
public databases and identified internally using genotyping-by-sequencing (GBS). A genetic map
with increased marker density was developed, and SrTA10187 was mapped to a 1.1 cM genetic
interval. To identify candidate resistance genes and to determine the genomic context
surrounding SrTA10187 two approaches were used. In the first approach, genetic markers were
aligned to the reference Aegilops tauschii genome developed by Jia et al. (2013). Due to large
reference genome recombination bins and conflicting marker orders, the genomic context
surrounding SrTA10187 could not be resolved. Alternatively, the use of a higher resolution Ae.
tauschii genetic map and pooled BAC library sequences developed by Luo et al. (2013) proved
to be more successful. After aligning common markers and annotating BAC library sequence in
the region surrounding SrTA10187, at least one NLR was identified in the interval of interest (of
the reference Aegilops tauschii accession). The marker resources and high resolution genetic
map developed in this study will facilitate continued efforts to improve stem rust resistance in
wheat using marker-assisted selection, and will enable gene pyramiding of SrTA10187 to
improve the durability of major-gene resistance.
Identification of the novel powdery mildew resistance gene Pm58 from Ae. tauschii
began by screening a small collection of Ae. tauschii accessions for seedling resistance using
detached-leaf assays. Of the nine accessions screened, TA1662 stood out with resistance to 18 of
the 20 isolates tested. After confirming that powdery mildew seedling resistance was segregating
as a single-locus trait in a population of wheat-Ae. tauschii introgression lines, the resistance

44

locus was mapped to wheat chromosome 2DS and formally designated Pm58. Genome-wide
genetic maps (including the resistance locus) were developed from GBS data, and tag sequences
were aligned to the reference Ae. tauschii genome to determine the chromosome identities of
linkage groups. To confirm the effectiveness of Pm58 in adult plant tissues, the same
introgression lines were rated for powdery mildew resistance in two naturally infected field
trials. In both environments, single major-effect QTLs were identified at the same locus where
Pm58 seedling resistance mapped. To improve the genetic map in the region immediately
surrounding Pm58, nine GBS-SNP markers were converted to KASP™ markers. Four KASP™
markers colocalized with Pm58 and will be useful for marker-assisted selection.
For either SrTA10187 or Pm58 to have an impact on wheat disease resistance breeding,
lines with acceptable agronomics that are fixed for the resistance locus must be accessible to the
public. To accomplish this for Pm58, two homozygous lines, U6714-A-011 and U6714-B-056,
were yield tested in multiple locations across the United States. Generally, U6714-B-056 had
higher yield potential than U6714-A-011, but both lines underperformed relative to locally
adapted check varieties and the recurrent wheat parent KS05HW14. To further characterize
isolate-specificity of Pm58, the two lines were inoculated with 20 powdery mildew isolates
collected throughout central- and eastern-United States. Isolate-specific interactions differed
slightly between U6714-A-011 and U6714-B-056, but both lines were more powdery mildew
resistant than the recurrent wheat parent KS05HW14. Seed of these germplasm lines has been
archived in the USDA-ARS National Center for Genetic Resources Preservation storage facility
in Fort Collins, CO, and active seed stocks will be maintained by the National Small Grains
Collection in Aberdeen, Idaho. Similar efforts to release SrTA10187 germplasm are underway

45

through a collaboration with the Hard Winter Wheat Genetics Research Unit, USDA-ARS, in
Manhattan, KS.

Overcoming past limitations in disease resistance breeding
Since the earliest attempts to map agronomically important traits in wheat, recombination
rate and marker availability have remained the primary limitations. Matters are further
complicated by introgression of alien loci that may not recombine readily with wheat and can be
linked to loci that reduce grain quality and yield (Wulff and Moscou 2014). This linkage drag
often hampers genetic gain for important breeding targets and discourages disease resistance
breeding. As demonstrated in the studies described here, development of wheat-Ae. tauschii
introgression lines with high background wheat isogeneity and using current SNP genotyping
platforms progress has been made towards overcoming some of these hurdles. By using the D
genome progenitor species Ae. tauschii, homologous D genome loci could recombine normally
and mapping efforts were simplified to diploid segregation of only D genome loci. Higher
throughput GBS and KASP™ marker platforms increased the speed and resolution of mapping.
With improved marker resolution and genetic maps, the location of disease resistance genes
could be more accurately defined and tightly linked genetic markers developed. Now efforts can
be refocused on reducing the genetic load of deleterious loci from Ae. tauschii, identifying welladapted germplasm, and deploying novel resistance genes in combination other major-gene or
adult-plant resistance to increase durability.

46

Future perspectives
In the past, well-funded plant breeding programs have engaged in positional cloning of
disease resistance genes. Now, the argument can be made that resistance gene cloning should be
practiced in specialized labs using advanced techniques that rely on variations of bulked
segregant analysis, chemical mutagenesis, or genomic reduction by resistance gene enrichment
(RenSeq) or chromosome flow sorting (Bent 2016, Sanchez-Martin et al. 2016). Two recent
studies relied on a RenSeq approach to preferentially capture and sequence DNA fragments
belonging to the NLR gene family (Jupe et al. 2013). In the first study, multiple lines were
chemically mutagenized to independently knock-out resistance gene function. Then, by
searching for a single resistance gene that was mutated in all the knock-out lines, the resistance
gene was cloned (Steuernagel et al. 2016). In the second study, after fine mapping the resistance
locus, RenSeq was used in combination with single-molecule real-time sequencing to de novo
assemble and clone six NLR genes in the region (Witek et al. 2016). Alternatively, genome
reduction has also been done using flow cytometry to isolate and sequence single chromosomes
linked to resistance. Again, by identifying contigs that were mutated in multiple knock-out lines,
the resistance gene was identified and cloned (Sanchez-Martin et al. 2016). Although none of
these techniques are the “silver bullet” for disease resistance gene cloning, they each represent
progress towards more cost-effective and less time-consuming methods. One caveat, however, is
that labs with established protocols, specialized equipment, and bioinformatics expertise will be
much better suited for the task.

47

APPENDIX

48

Characterizing YrTA1718 from Aegilops tauschii in hexaploid wheat and germplasm
development

Introduction
Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), has become one of
the most problematic diseases affecting wheat (Triticum aestivum L.; Schwessinger 2017). In the
last 50 years, Pst virulence and race diversity has gradually increased with the introduction of
major gene resistance in wheat (Liu et al. 2017). Starting around the year 2000, an unprecedented
rise in Pst virulence and disease severity was observed in the United States, Australia, and
Europe (Hovmoller et al. 2010, Hubbard et al. 2015). This recent trend is especially concerning
in the eastern United States soft wheat region where stripe rust was not problematic in the past
and most commercial wheat varieties are Pst-susceptible (Cereal Rust Bulletins,
www.ars.usda.gov). Due to recent stripe rust epidemics, wheat breeders are rapidly searching for
effective sources of resistance.
There is a long tradition of using wild wheat relatives, including the diploid D genome
progenitor species Aegilops tauschii Coss., as reservoirs for novel disease resistance genes.
Although numerous attempts have been made to transfer stripe rust resistance from Ae. tauschii
to hexaploid wheat, only one formally designated Ae. tauschii resistance gene, Yr28, has been
mapped in wheat (Singh et al. 2000). Another temporarily designated resistance gene, YrAS2388
(also derived from Ae. tauschii), was mapped to a 4DS locus near Yr28 and expressed different
Pst race-specificity (Huang et al. 2011). Many other attempts to transfer resistance from Ae.
tauschii to wheat were less successful due to suppression of stripe rust resistance in hexaploid
genomic backgrounds (Ma et al. 1995, Yang et al. 2003, Chen et al 2013).

49

A small population of BC2F4-derived wheat introgression lines (U6719) was developed
by direct hybridization and backcrossing of the stripe rust resistant Ae. tauschii accession
TA1718 and the susceptible wheat line KS05HW14. The objectives of this study were to
characterize seedling and adult plant resistance segregating in U6719, and to develop improved
germplasm and F2 mapping populations. Additionally, the resistance from TA1718 will be
integrated into the soft white winter wheat variety Ambassador using backcross-breeding to
improve stripe rust resistance resources in the eastern US soft wheat growing region.

Methods and Materials
Plant materials
The stripe rust resistant Ae. tauschii accession TA1718 was originally collected in Iran
and is currently maintained by the Wheat Genetic and Genomic Resource Center in Manhattan,
KS (www.genesys-pgr.org). TA1718 was hybridized directly with the hard white winter wheat
line KS05HW14 (Figure 5.1). Interspecific F1 embryos were rescued on growth media following
the method described by Olson et al. (2013). Recovered F1 plants were used as females in an
initial backcross to the recurrent wheat parent KS05HW14. A single BC1F1 plant was recovered
and backcrossed to KS05HW14 as the male parent. A total of 15 BC2F1 plants belonging to the
U6719 family were advanced by single-seed-descent to the BC2F4 generation when the seed
from a single plant was harvested and increased in subsequent generations to produce BC2F4derived lines.
The wheat introgression line U6719-004 was selected for mapping population
development following stripe rust resistance screening and yield testing (Figure 5.1). U6719-004
was backcrossed as a male with KS05HW14, and BC3F1 plants were self-fertilized to establish a

50

Figure 5.1 Introgression of stripe rust resistance from Ae. tauschii accession TA1718 and
mapping population development. In hybridization diagrams, female parents are listed on the
left-side. Encircled X indicates self-fertilization.

51

BC3F2 mapping population. Likewise, U6719-004 was crossed as a male with the soft white
winter wheat variety Ambassador, and F1 plants were self-fertilized to establish an additional F2
mapping population. Ambassador is a high yielding, stripe rust susceptible wheat line that is
well-adapted to Michigan growing environments.

Seedling stripe rust evaluation
Seedling stripe rust phenotyping of BC2F4-derived wheat lines and F2 mapping
populations was done in the growth chamber facility at Michigan State University. Stripe rust
urediniospores used in this study were isolated from naturally occurring infections in Michigan
and confirmed to be race Pstv-37 based on reactions to a set of single-gene differential wheat
lines (Wan and Chen 2014). Following a 5 min heat-shock at 45°C, urediniospores were
suspended in Soltrol 170 isoparaffin oil (Chevron Philips Chemical Company LP, The
Woodlands, TX) and sprayed onto two-leaf seedlings using an airbrush. Plants were then
incubated in a dew chamber at 14°C and 100% relative humidity for 18 h. After incubation,
plants were transferred back to a growth chamber held at 14°C. Seedling infection types (IT)
were recorded at 18 days post inoculation using a 0 to 9 scale described previously (Wan and
Chen 2014).

Adult plant stripe rust evaluation and yield testing
Adult plant stripe rust phenotyping of U6719-004, U6719-009, and the recurrent parent
KS05HW14 was done at six locations across the United States in the year 2016. All the locations
included in this study experienced near-epidemic levels of stripe rust in 2016 due to a mild
winter followed by a cool spring which lead to early and severe onset of disease. Each location

52

was naturally infected by Pst races present in the region at that time. Four of the six locations
were planted as yield trials: Brookings, SD; Hays, KS; Pullman, WA; and Richville, MI. The
remaining two locations, Central Ferry, WA and Fayetteville, AR, were planted as head rows.
Flag leaf severity was rated at all locations as the percentage (0-100%) of leaf area covered with
stripe rust urediniospores (Peterson et al. 1948). At four locations, the Pst IT (0-9) was also rated
on flag leaves (Wan and Chen 2014).
Yield testing of U6719-004, U6719-009, KS05HW14, and locally adapted check
varieties was conducted at ten year-by-location environments. Lines were tested in two
consecutive years (2015 and 2016) at Ashland, KS; Hays, KS; and Richville, MI. The remaining
locations, Brookings, SD; Champaign, IL; Marianna, AR; and Pullman, WA, were only tested
once in 2016. The yield trial locations are the same as those where adult plant stripe rust was
rated. At each trial location, U6719-004 and U6719-009 were planted in single replicate plots in
an augmented design containing six incomplete blocks. Locally adapted check varieties and the
recurrent parent KS05HW14 were replicated in each block and used for yield comparisons and
block corrections. Using a mixed linear model where genotype was treated as a fixed effect and
block was treated as a random effect, grain yield least squares means (LS-means) and 95%
confidence intervals were calculated in Rstudio® (RStudio, Boston, MA, USA, R version 3.2.1)
using the packages lme4 (v.1.1-12) and lsmeans (v.2.25-5).

Preliminary Results
Identification of stripe rust resistance from TA1718
A small population of BC2F4-derived wheat introgression lines, U6719 (n = 15), was developed
from the direct hybridization of the stripe rust resistant Ae. tauschii accession TA1718 and

53

susceptible wheat line KS05HW14. To ascertain if resistance from TA1718 was successfully
transferred and expressed in a hexaploid wheat background, U6719 wheat introgression lines
were screened for seedling stripe rust resistance. A total of 4 plants expressed seedling resistance
to Pstv-37 and had an IT ranging from 3-4 (Table 5.1 and 5.2). The segregation ratio of resistant
to susceptible plants indicates that stripe rust resistance segregated as a single-locus trait (Table
5.1, χ2 = 0.02, P-value = 0.88) and was temporarily designated YrTA1718.

Characterizing adult plant resistance and yield in U6719-004 and U6719-009
Two BC2F4-derived wheat introgression lines, U6719-004 and U6719-009, confirmed to
be stripe rust resistant by seedling tests were selected for adult plant stripe rust resistance
characterization. In 2016, both lines were evaluated in six diverse United States environments
including: Brookings, SD; Central Ferry, WA; Fayetteville, AR; Hays, KS; Pullman, WA; and
Richville, MI. Naturally occurring stripe rust incidence and severity was high across all
locations. U6719-004 and U6719-009 expressed adult plant resistance in every environment
tested and had consistently lower IT and flag leaf severity compared to the recurrent parent
KS05HW14 (Table 5.2). Variation in IT and severity scores between locations may be accounted
for by differences in Pst race profiles.
The same two lines were also yield tested in 10 year-by-location environments between
2015 and 2016. Yield trial locations included: Ashland, KS; Brookings, SD; Champaign, IL;
Hays, KS; Marianna, AR; Pullman, WA; and Richville, MI. U6719-004 and U6719-009 both
exhibited exceptional yield performance relative to the recurrent wheat parent KS05HW14
(Table 5.3). In six out of ten environments, U6719-004 had higher yield than mean KS05HW14
yield. In five out of ten environments, U6719-009 had higher yield than mean KS05HW14 yield.

54

Table 5.1 Segregation of Pst resistance in BC2 F4 -derived introgression lines and BC3 F2 and F2 mapping populations
Number of plants
Family
Pedigree
Generation
a
Susceptible
Resistant
11
4
U6719
KS05HW14///KS05HW14/TA1718//KS05HW14 BC2 F4:6

Expected ratio
Total
15

(R:S)b
1:3

χ2

P -value

0.02

0.88

MSU16000953 KS05HW14/U6719-004

BC3 F2

34

72

106

3:1

104.16

<0.001

MSU16000955 Ambassador/U6719-004

F2

38

61

99

3:1

70.79

<0.001

a

Plants with an infecgtion type < 7 were classified resistant

b

Assuming YrTA1718 dominance

55

Table 5.2 Seedling and adult Pst infection types and severity on wheat lines KS05HW14, U6719-004, and U6719-009.
KS05HW14
U6719-004
U6719-009
Location
Year
Growth stage
IT (0-9)
Severity
IT (0-9)
Severity
IT (0-9)
Severity
Growth Chamber (race Pstv-37 )
Brookings, SD (yield trial)
Central Ferry, WA (head rows)

2015
2016
2016

Seedling
Adult
Adult

a

8

7

3

-

4

-

a

-

16%

-

7%

a

41%

a

3

25%

3

20%

a

-

a

7%

-

5%

a

3

10%

1

5%

a

5

30%

6

40%

a

3

10%

3

10%

73%

Fayetteville, AR (head rows)

2016

Adult

-

59%

Hays, KS (yield trial)

2016

Adult

9a

74%

Adult

a

Pullman, WA (yield trial)
Richville, MI (yield trial)
a

2016
2016

Adult

8

a

7

48%
32%

Mean IT and severity reported for replicate growth chamber pots or field plots. Resistant infection types are indicated in bold.

56

a

Table 5.3 Grain yield LS-means of locally adapted check varieties, KS05HW14, U6719-004, and U6719-009 in 10 diverse environments throughout the United States.
-1
 
 
 
---------------------------------------------------------------t ha -------------------------------------------------------------------_____Locally adapted check varieties_____ _____KS05HW14_____
U6719-004
U6719-009
Grain yield
95% CI
Grain yield
95% CI
Grain yield (% KS05HW14) Grain yield (% KS05HW14)
Location
Year
Name
2015
Everest
4.64
4.45 - 4.82
4.66
4.41 - 4.89
4.99 (107%)
5.04 (108%)
Ashland, KS
2016
Everest
4.85
4.65 – 5.05
4.2
4.11 – 4.30
4.01 (95%)
3.37 (80%)
Ashland, KS
3.6
3.30 - 3.90
3.3
3.17 - 3.44
5.39 (163%)
5.32 (161%)
Brookings, SD 2016
Lyman
Champaign, IL 2016
IL07-19334
7.85
7.47 - 8.22
5.67
5.50 - 5.85
4.57 (81%)
4.26 (75%)
3.05 - 3.40
4.02 (125%)
3.62 (112%)
Hays, KS
2015
Ernie
2.73
2.34 - 3.12
3.22
5.25 (145%)
3.61
3.56 - 3.66
4.69 (130%)
Hays, KS
2016
Joe
6.76
6.65 - 6.87
AR11LE24
4.12
3.86 - 4.39
2.73
2.60 - 2.85
2.86 (105%)
3.01 (110%)
Marianna, AR 2016
4.45
4.31 - 4.59
4.08 (92%)
3.63 (82%)
Pullman, WA
2016
Jasper
9.06
8.75 - 9.37
2015
5.09 - 5.26
4.95
4.76 - 5.15
4.48 (91%)
4.43 (89%)
Richville, MI
AC Mountain
5.18
6.13 (115%)
Richville, MI
2016
Ambassador
5.63
5.31 - 5.95
5.31
5.15 - 5.46
5.03 (95%)
CI = confidence interval.

57

In a few locations, U6719-004 and U6719-009 even outperformed locally adapted check
varieties. It is likely that stripe rust resistance fixed in U6719-004 and U6719-009 contributed to
yield performance, especially when compared to susceptible check varieties including
KS05HW14.

Mapping population seedling resistance
With the intent to eventually map YrTA1718, U6719-004 was crossed with KS05HW14
and the Pst-susceptible soft white wheat variety Ambassador to develop BC3F2 and F2 mapping
populations, respectively (Figure 5.1). Using the race Pstv-37, 106 BC3F2 and 99 F2 plants were
screened for seedling stripe rust resistance. In both populations, resistance was expressed in
approximately 35% of individuals which did not conform to the expected segregation ratio of a
dominant resistance gene (Table 5.1, BC3F2 χ2 = 104.16, P-value <0.001 and F2 χ2 = 70.79, Pvalue <0.001). Although the resistance response was not as complete as that of the resistance
donor Ae. tauschii accession TA1718, the resistant phenotype was evident from extensive leaf
chlorosis and necrosis and reduced Pst sporulation. Susceptible BC3F2 and F2 lines resembled the
susceptible wheat parents KS05HW14 and Ambassador (Figure 5.2).

Discussion
The successful introgression of YrTA1718 from Ae. tauschii into wheat was demonstrated
by seedling resistance screening of BC2F4-derived wheat introgression lines. Two lines fixed for
YrTA1718 seedling resistance also expressed adult plant resistance at field sites throughout the
United States—which may indicate that YrTA1718 is effective against a broad range of Pst races

58

Figure 5.2 Pstv-37 seedling leaf infection types of KS05HW14, TA1718, Ambassador, and
resistant and susceptible BC3F2 and F2 individuals.

59

(Annual Stripe Rust Race Reports, http://striperust.wsu.edu/races/data/). The importance of
stripe rust disease resistance was also highlighted by higher grain yield of resistant lines U6719004 and U6719-009 compared to the susceptible parent KS05HW14 at locations with high
disease pressure. In fact, either line may be suitable for immediate germplasm release.
Ongoing efforts to map YrTA1718 and backcross-breed resistance into a soft white winter
wheat background were initiated by the development of BC3F2 and F2 mapping populations. It is
still unknown if YrTA1718 is a novel disease resistance gene, or if it is one of the previously
identified Ae. tauschii resistance genes Yr28 or YrAS2388. Mapping of YrTA1718 will help
elucidate its relationship to other known resistance genes and inform breeding decisions.
Resistant F2 progeny were recovered from a cross between U6719-004 and Ambassador which
indicates that YrTA1718 was not suppressed. Additional backcrossing with Ambassador and
resistance selection should be continued to develop a stripe rust resistant soft white winter wheat
line that is well adapted to the eastern US soft wheat growing region (Figure 5.1).
Finally, based on the segregation ratio of resistant and susceptible plants in BC3F2 and F2
mapping populations it appears that YrTA1718 is not a dominant resistance gene. An alternative
hypothesis that may explain the unexpected segregation ratio could be that YrTA1718 is a
recessive resistance gene, or that deleterious alleles from Ae. tauschii caused segregation
distortion. It is also possible that YrTA1718 behaves in a dosage-dependent manner, where
heterozygous individuals express a more susceptible response that is hard to distinguish from
homozygous susceptible individuals. Genetic markers linked to YrTA1718 and progeny tests of
BC3F2:3 and F2:3 plants will likely clarify this observation.

60

REFERENCES

61

REFERENCES

Bent A (2016) Resistance from relatives. Nat. Biotechnol. 34(6):620-621
Chen W, Liu T, Gao L (2013) Suppression of stripe rust and leaf rust resistances in interspecific
crosses of wheat. Euphytica 192:339-346. doi: 10.1007/s10681-012-0854-2
Huang L, Zhang LQ, Liu BL, Yan ZH, Zhang B, Zhang HG, Zheng YL, Liu DC (2011)
Molecular tagging of a stripe rust resistance gene in Aegilops tauschii. Euphytica
179:313-318. doi: 10.1007/s10681-010-0330-9
Hubbard A, Lewis CM, Yoshida K, Ramirez-Gonzalez RH, de Vallavieille-Pope C, Thomas J,
Komoun S, Bayles R, Uauy C, Saunders DGO (2015) Field pathogenomics reveals the
emergence of a diverse wheat yellow rust population. Genome Biol. 16:23. doi:
10.1186/s13059-015-0590-8
Jia J, Zhao S, Kong X, Li Y, Zhao G, He W, Appels R et al. (2013) Aegilops tauschii draft
genome sequence reveals a gene repertoire for wheat adaptation. Nature 496:91-95. doi:
10.1038/nature12028
Jupe F, Witek K, Verweij W, Sliwka J, Pritchard L, Etherington GJ, Maclean D, Cock PJ,
Leggett RM, Bryan GJ, Cardle L, Hein I, Jones JDG (2013) Resistance gene enrichment
sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced
plant genomes and rapid mapping of resistance loci in segregating populations. Plant J.
76:530-544. doi: 10.1111/tpj.12307
Liu T, Wan A, Liu D, Chen X (2017) Changes of races and virulence genes in Puccinia
striiformis f. sp. tritici, the wheat stripe rust pathogen, in the United States from 1968 to
2009. Plant Dis. doi: 10.1094/PDIS-12-16-1786-RE
Luo MC, Gu YQ, You FM, Deal KR, Ma Y, Hu Y, Juo N, Wang Y, Wang J, Chen S, Jorgensen
CM, Zhang Y, McGuire PE, Pasternak S, Stein JC, Ware D, Kramer M, McCombie WR,
Kianian SF, Martis MM, Mayer KFX, Sehgal SK, Li W, Gill B, Bevan MW, Simkova H,
Dolezel J, Weining S, Lazo GR, Anderson OD, Dvorak J (2013) A 4-gigabase physical
map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the
wheat D-genome progenitor. PNAS 110(19):7940-7945.
Ma H, Sing RP, Mujeeb-Kazi A (1995) Suppression/expression of resistance to stripe rust in
synthetic hexaploid wheat (Triticum turgidum x T. tauschii). Euphytica 83:87-93
Olson EL, Rouse MN, Pumphrey MO, Bowden RL, Gill BS, Poland JA (2013) Simultaneous
transfer, introgression, and genomic localization of genes for resistance to stem rust race
TTKSK (Ug99) from Aegilops tauschii to wheat. Theor. Appl. Genet. 126:1179-1188.
doi: 10.1007/s00122-013-2045-5
62

Peterson RF, Campbell AB, Hannah AE (1948) A diagrammatic scale for estimating rust
intensity on leaves and stems of cereals. Can. J. Res. 26c(5):496-500. doi:
10.1139/cjr48c-033
Sanchez-Martin J, Steuernagel B, Ghosh S, Herren G, Hurni S, Adamski N, Vrana J, Kubalakova
M, Krattinger SG, Wicker T, Dolezel J, Keller B, Wulff BH (2016) Rapid gene isolation
in barley and wheat by mutant chromosome sequencing. Genome Biol. 17:221. doi:
10.1186/s13059-016-1082-1
Schwessinger B (2017) Fundamental wheat stripe rust research in the 21st century. New Phytol.
213:1625-1631. doi: 10.1111/nph.14159
Sing RP, Nelson JC, Sorrells ME (2000) Mapping Yr28 and other genes for resistance to stripe
rust in wheat. Crop Sci. 40:1148-1155
Steuernagel B, Periyannan SK, Hernandez-Pinzon I, Witek K, Rouse MN, Yu G, Hatta A,
Ayliffe M, Bariana H, Jones JDG, Lagudah ES, Wulff BBH (2016) Rapid cloning of
disease-resistance genes in plants using mutagenesis and sequence capture. Nature
Biotechnol. 34(6):652-655. doi: 10.1038/nbt.3543
Wan AM, Chen XM (2014) Virulence Characterization of Puccinia striiformis f. sp. tritici using
a new set of Yr single-gene line differentials in the United States in 2010. Plant Dis.
98:1534-1542. doi: 10.1094/PDIS-01-14-0071-RE
Witek K, Jupe F, Witeck AI, Baker D, Clark MD, Jones JDG (2016) Accelerated cloning of
potato late blight-resistance gene using RenSeq and SMRT sequencing. Nature
Biotechnol. 34(6):656-660. doi: 10.1038/nbt.3540
Wulff BBH, Moscou MJ (2014) Strategies for transferring resistance into wheat: from wilde
crosses to GM cassettes. Front. Plant Sci. 5:692. doi: 10.3389/fpls.2014.00692
Yang WY, Yu Y, Zhang Y, Hu XR, Wang Y, Zhou YC, Lu BR (2003) Inheritance and
expression of stripe rust resistance in common wheat (Triticum aestivum) transferred
from Aegilops tauschii and its utilization. Hereditas 139:49-55

63