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Genetic analysis and molecular mapping of

resistance to Puccinia striiformis f. sp. pseudo-
hordei in common wheat

Article in Plant Pathology · July 2016

DOI: 10.1111/ppa.12580

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 Received Date : 12-May-2016
Accepted Article
Revised Date : 21-Jun-2016

Accepted Date : 21-Jun-2016

Article type : Original Article

Genetic analysis and molecular mapping of resistance to Puccinia striiformis f. sp.

pseudo-hordei in common wheat

Dracatos PM*, Haghdoust R, Park RF

The University of Sydney, Plant Breeding Institute, Cobbitty, Private Bag 4011, Narellan, NSW, 2567,

Australia.

* Email: peter.dracatos@sydney.edu.au (corresponding author-Dracatos)

Abstract

This is the first genetic study reporting on the interaction and molecular mapping of

resistance to the barley grass stripe rust pathogen (Puccinia striiformis f. sp. pseudo-hordei

– Psph) in common wheat. We tested 638 wheat accessions as seedlings and determined

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 that wheat is a near-nonhost to Psph based on rare susceptibility observed in <2% of
Accepted Article
commercial cultivars and <5% of wheat landraces. As previously observed for P. s. f. sp.

tritici – Pst the Australian cultivar Teal was highly susceptible to Psph. In contrast, a

selection of cv. Avocet carrying complementary resistance genes Yr73 and Yr74 (Avocet

R; AvR) was resistant. We used the Teal/AvR (T/A) doubled haploid (DH) population to

map resistance in AvR to Psph. Infection types on the T/A DH lines inoculated with Psph

and Pst indicated that all DH lines carrying both Yr73 and Yr74 were also resistant to

Psph, however, fewer DH lines were susceptible to Psph than expected suggesting the

resistance was more complex. Both QTL and marker-trait association (MTA) analysis

using 9,053 DArT-Seq markers determined that resistance to Psph was polygenically

inherited and mapped to chromosomes 3A, 3D, 4A and 5B. The 3DL and 5BL markers

co-located with Yr73 and Yr74 suggesting an overlap between host and non-host

resistance mechanisms.

Keywords: barley grass stripe rust, near-nonhost, resistance

Introduction

Stripe rust, caused by P. striiformis Westend, is one of the most important and damaging

foliar diseases of both crop and grass species within the Pooideae subfamily of the

Poaceae (Wellings 2007; 2011). Wheat stripe rust (P. striiformis f. sp. tritici - Pst) and

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 barley stripe rust (P. striiformis f. sp. hordei -Psh) are among the most important diseases
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infecting common bread and durum wheat, triticale and barley, respectively (Wellings

et al., 2000a; Wellings 2007; 2011). In 1997, a variant of P. striiformis was identified on a

wild Hordeum species (Hordeum leporinum- locally known as barley grass) in Australia

(Wellings et al., 2000b). The pathogen was avirulent on all of the International, European

and Australian wheat stripe rust differential lines, with the exception of partial

virulence for Chinese 166 (carries resistance gene Yr1) wheat, a feature typical of the

exotic barley stripe rust pathogen Psh (Johnson et al., 1972; Stubbs 1985; Line and

Qayoum 1991; Wellings et al., 2000b). Subsequent phenotypic testing of Australian

barley cultivars determined that approximately 90% were resistant to Psph in contrast to

the high frequency of susceptibility observed in adult plant testing of the same cultivars

at Toluca in Mexico with Psh race 24 (Wellings et al., 2000a). The variant was described

as a new forma specialis, P. striiformis f. sp. pseudo-hordei (Psph) (Wellings 2007), based on

pathogenicity and molecular marker data that grouped Psph in a taxonomic clade

distinct from both Pst and Psh (Bailey et al., 2013).

Psph is currently not a serious threat to the Australian barley industry. Previous

reports of somatic hybridization (Park and Wellings 2012), step-wise mutation

(Wellings and McIntosh 1990; Wellings 2007) and exotic migration (Watson and de

Sousa 1982; Hovmøller et al., 2002) in cereal rust pathogens including P. striiformis

however, suggest that there is a potential for Psph to acquire virulence for commonly

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 deployed resistance genes in commercial wheat and barley cultivars. Sanghi (1968)
Accepted Article
reported virulence to the wheat stem rust resistance genes Sr5 and Sr11 in the rye stem

rust pathogen P. graminis f. sp. secalis- Pgs and other somatic and sexual hybrids of P.

graminis.

The immunity of an entire plant species against all genotypes of a

phytopathogen is defined as nonhost resistance. The genetic basis of nonhost resistance

in cereal crop species to rust pathogens is by definition challenging due to the

requirement of susceptible genotypes for genetic studies. There are some plant-

pathogen combinations where only 10% or less of accessions are susceptible, these plant

species have been referred to as near-nonhosts or intermediate hosts (Atienza et al.,

2004). The availability of occasional susceptible plant genotypes permits investigation of

the genetic basis of host-status to specialized phytopathogens. Despite being classified

as a distinct forma specialis, the host range and specificity of Psph on different grass and

crop species of the Triticeae is poorly understood. Very early studies by Sanghi (1968)

investigated the inheritance of resistance in wheat to the non-adapted (heterologous)

rye stem rust pathogen Pgs and other P. graminis cultures with unusual avirulence on

common wheat. These studies determined that the resistance in two wheat cvs., Pusa

and Mona, was simply inherited and due to four and three genes, respectively.

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 To date, three genetic studies have examined inheritance of resistance in barley
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to Psph. Golegaonkar et al., (2013) mapped a single dominant resistance gene in the

barley landrace ‘Sahara’ on chromosome arm 7HL; Derevnina et al., (2015) mapped

single resistance genes in Yerong to chromosome 5HL and in Franklin to chromosome

7HL and Kamino et al., (2016) identified major-effect resistance QTLs on chromosomes

1HS and 7HL, derived from the barley genotypes WI2585 and Haruna Nijo,

respectively. Currently, more than 74 loci conferring resistance to Pst have been

designated (Yr1- Yr74; Yr26 is considered to be synonymous with Yr24), predominately

from common wheat but also from durum wheat and wild progenitors (Park 2015;

Dracatos et al., 2016). A selection of the Australian wheat cultivar Avocet (AvR) carries

complementary genes (Yr73 and Yr74) for seedling resistance to Pst (Dracatos et al.,

2016). One of the genes was located to chromosome 3DL by Bariana (1991), and a recent

study mapped both genes to chromosomes 3DL and 5BL in a doubled haploid (DH)

population based on the genotypes Avocet R and Teal (Dracatos et al., 2016). Avocet R

(AvR) is also resistant to Psph whereas Teal is susceptible. We used the same DH

population and a diverse collection of wheat accessions to both map the resistance to

Psph in AvR and characterize the host status of Psph on common wheat.

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 Materials and methods
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Plant materials

A collection of 636 wheat accessions from USDA comprising diverse landraces (245)

and cultivated winter and spring wheats derived from Canada (86), Australia (121) and

Europe (186) were assessed for response to Psph (Supplementary Table 1). The

previously developed Teal/AvR wheat DH population (Dracatos et al., 2016) was used

in this study to assess the genetic basis of resistance to Psph in selection AvR. In brief,

the hexaploid spring wheat line AvR (carries the complementary resistance loci Yr73

and Yr74 – Dracatos et al., 2016) was crossed with the Australian spring wheat cultivar

Teal (AUS 12330), and a DH population consisting of 126 lines was produced.

Pathogen isolates

A single pustule (SP) isolate of Psph (isolate 981549: accession number, 589) was

generated from a Psph sample isolated from infected barley grass in Victoria in 1998.

This SP was multiplied on barley cultivar (cv.) Maritime (resistant to Pst and

moderately susceptible to Psph) in an isolated greenhouse compartment. The inoculum

was then checked for purity on a wheat stripe rust differential set and the infection

types (ITs) were compared in the same experiments to two Pst pathotypes that were

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 avirulent with respect to pathogenicity on the Avocet R (Yr73+Yr74) resistance (108
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E141 A- and 104 E137 A-) and a single virulent Pst pathotype, 108 E141 A+ (Table 1). A

UK-derived isolate of Psh (83.39, virulent on barley differentials Astrix, Bigo, Sultan,

Berac, Verunda and Atem), was used to phenotype selected T/A DH wheat lines at the

National Institute of Agricultural Botany (NIAB) in Cambridge, United Kingdom that

either carried (Yr73+Yr74) or lacked (either Yr73 only, Yr74 only, or lacking both genes)

the complementary resistance genes.

Seedling inoculation procedures

Seedlings (10 seedlings per experimental line) were grown to expose both the first and

second leaves in 9-cm plastic pots (two lines/pot) containing a potting mix comprising

composted pine bark and coarse sand in a ratio of 4:1. Pots were fertilized with a

soluble nitrogenous fertilizer Aquasol® (Hortico Pty Ltd, Australia) at the rate of 30 g in

10 L of water for 200 pots prior to sowing. Plants were maintained both before and after

inoculation in a temperature- controlled greenhouse (17-18°C) under high natural light

(>300 µEs-1m-1) as described by Wellings et al., (1988). Approximately 20 mg of

urediniospores was suspended in 10 ml of light mineral oil (IsoparL®, Univar, NSW,

Australia) and sprayed with a mist atomizer over the top of seedlings. Inoculated

seedlings were incubated overnight at 9–12°C in an enclosed chamber in the dark. After

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 incubation the seedlings were transferred to naturally lit microclimate rooms, where
Accepted Article
temperatures were maintained at 16–20°C. All experiments were repeated twice at

independent time-points using the same parameters.

Disease assessment

Disease response was assessed 16–18 days post inoculation, using a 0–4 infection type

(IT) scale as described for Pst by McIntosh et al., (1995). Where ’0’= no visible symptoms,

“;”= necrotic flecks, “;N”= necrotic areas without sporulation, “1”= necrotic and

chlorotic areas with restricted sporulation; “2”= moderate sporulation with necrosis and

chlorosis, “3” sporulation with chlorosis, “4” abundant sporulation without chlorosis.

Variations of the ITs were indicated by use of − (less than average for the class), + (more

than average for the class), C (chlorosis) and/or N (necrosis). Where plants with two or

more distinctly different low ITs were observed, data were recorded and separated with

a comma (e.g. IT=”1,1+” and IT=”3,3+”). Plants with ITs of “3” or higher were =

considered to be susceptible (McIntosh et al., 1995). The presence of chlorosis (C) or

necrosis (N) when distinctive was incorporated into the numerical (“0 – 4”) score

described by McIntosh et al., (1995). IT data was converted to a numerical 0-4 scale for

QTL mapping and modifiers to the IT such as + and - were incorporated by the addition

or subtraction of 0.25 (i.e. +) and 0.5 (i.e. ++).

Genotypic analysis and linkage map construction

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 The same DArT-Seq marker genotypic dataset and linkage map used to map the
Accepted Article
complementary resistance genes Yr73 and Yr74 from Avocet R (Dracatos et al., 2016)

was used in this study to map the resistance to Psph (data available on request). Whole-

genome profiling was performed on 86 of the 126 T/A DH lines using DArT-Seq™

technology by Diversity Arrays Technology Pty Ltd, Australia, as described by Killian

et al., (2012) and Raman et al., (2014). The chromosomal designation of each linkage

group was determined by the position of each DArT-Seq marker relative to the

corresponding position on the wheat DArT consensus genetic map and the wheat

genome pre-assembly data for each of the seven homeologous chromosome groups of

bread wheat.

Quantitative Trait Mapping (QTL) analysis

QTL analysis was performed on 86 T/A DH lines based on a previously published

genetic map build (Dracatos et al., 2016). Subsets of marker loci were selected to provide

even coverage of the genome with marker intervals of approximately 6 cM. Single

marker regression (SMR) was used initially to identify significant associations with

selected genetic markers. Composite Interval Mapping (CIM) methods were used to

identify and confirm the presence of QTL using the Multiple Trait Mapping algorithm

in QTL Cartographer 2.5 software (Wang et al., 2012). The maximum LOD score of

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 association between the genotype and trait data was calculated for CIM, and QTL
Accepted Article
location predictions were accepted for CIM for values greater than a threshold value of

3. Permutation analysis (1,000 iterations) was used to establish an experiment-wise

significance value at the 0.05 confidence level, defined as a minimum LOD threshold for

each trait in CIM (Churchill and Deorge 1994; Doerge and Churchill 1996). For CIM

analysis, the maximum LOD value, additivity and the extent of drops by 1 and 2 LOD

units from the maximum value on the genetic map were tabulated.

Results

Determining the host status of common wheat to Psph

Psph was partially virulent on the wheat stripe rust seedling resistance gene, Yr1

compared to the avirulence observed using Pst pathotype 108 E141 A- and avirulent on

the remaining Pst differentials including Avocet S, indicating that the isolate used for

phenotypic analysis was not contaminated with Pst (Table 1). Of the 638 wheat

accessions (393 modern cultivars and 245 landraces) tested, less than 2% of commercial

cultivars and <5% of wheat land races were susceptible to Psph at the seedling stage

(Fig. 1A, Fig 2A and Supplementary Table 1). Furthermore, sporulation occurred on

only 3.3% of the cultivated wheats, compared with 10.2% of the landraces, suggesting

wheat is probably a near-nonhost species to the heterologous P. striiformis pathogen,

Psph (Fig. 1A, Fig 2A and Supplementary Table 1). More cultivated wheats were

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 immune to Psph relative to landraces, however more of the landraces displayed
Accepted Article
intermediate resistance with either necrosis or chlorosis (Fig. 1A, Fig 2A and

Supplementary Table 1).

Response of Teal/AvR population to Psph and Psh

As previously observed for the wheat stripe rust pathogen Pst, the Australian cultivar

Teal was susceptible to Psph at the seedling stage (IT = “3++”) (Fig. 1, Fig. 2B, Table 2

and Supplementary Table 2). In contrast, AvR carrying the complementary seedling

resistance genes Yr73 and Yr74 (previously referred to as YrA) was resistant (IT= “;C”)

to Psph (Fig. 1B. and Fig. 2B). Without exception, all DH lines carrying both

complementary genes (Yr73+Yr74) were resistant to Psph (Supplementary Table 2). ITs

observed on DH lines with Yr73+Yr74 were also consistently lower (“0;” to “;C”)

compared to those observed in response to Pst pt. 108 E141 A- (“;C” to “23C”) (Table 2;

Supplementary Table 2). Single gene stocks with the individual complementary

resistance genes Yr73 (T/A63) and Yr74 (T/A72) identified previously as seedling

susceptible to Pst pathotype 108 E141 A- were both intermediate (IT = “2+C to 3C”) in

response to Psph (Fig. 1B, Supplementary Table 2). Both parental genotypes (AvR and

Teal) and selected DH lines postulated either to carry or lack Yr73+Yr74 were also

assessed in the UK with isolate Psh83.39 of the heterologous barley stripe rust pathogen

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 (Psh) (Table 2). All Yr73+Yr74 positive lines tested had lower ITs than those lacking both
Accepted Article
genes. T/A45 (no resistance to Pst) was the most susceptible line (“12+NN”), relative to

other DH lines tested carrying the complementary resistance to Pst (Yr73 + Yr74) (Table

2). While susceptible to Psph, Teal was resistant to Psh (infection type “;CN”). Lines

AvR, DH13 and DH77 (Yr73+Yr74, +) all produced lower ITs (“;C”) in response to Psh

relative to DH lines lacking Yr73+Yr74 (Table 2).

Genetic map construction of the T/A DH mapping population

The genotypic data of same T/A DH mapping population previously used to map

complementary genes (Yr73 and Yr74) (Dracatos et al., 2016) was also used to map the

resistance in AvR to Psph in this study (Table 3, Fig. 3, ). The T/A genetic map contained

9053 polymorphic markers spanning 34 linkage groups. In a number of instances

chromosomes with more than one linkage group were identified. This was attributed to

situations when there were no markers in a region of high recombination and the

recombination measured between the markers on either side was larger than the

threshold used for linkage group binning (Supplementary Table 3, Supplementary Fig.

1). The classification of each linkage group to specific chromosomal arms was

performed based on similarity to the wheat genome preassembly and the genetic

position of each DArT marker relative to the wheat DArT consensus map

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 (Supplementary Table 3). The genetic map of the T/A DH population was also found to
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carry at least two independent translocations (Supplementary Fig. 1). A large

translocation on chromosome 6AL from Thinopyrum ponticum (carrying the wheat stem

rust resistance gene Sr26) contributed by Avocet R parent explains the large linkage

block on chromosome 6A in the T/A genetic map. The second is the widely reported

5B:7B reciprocal translocation derived from the Teal parent was also identified in the

genetic map of the T/A DH population and is known to carry one of the complementary

resistance genes to stripe rust Yr74 (Dracatos et al., 2016).

QTL analysis of resistance to Psph in the T/A wheat mapping population

The phenotypic variation for each of the experimental replicates was not bimodal and

was skewed towards resistance, suggestive of polygenic inheritance (Fig. 2B). Single

marker regression (SMR) analysis was performed between phenotypic scores for both

replicates and marker genotypic data and in all instances significantly associated (P <

0.001) marker loci were exclusively located in chromosomal regions containing QTL

(Table 3). Multiple trait mapping (MTM) identified nine chromosomal regions across

both experimental replicates that were associated with resistance to Psph (Rpsphq1-9)

(Supplementary Fig. 2), however only five resistant loci (Rpsphq1, 2, 3, 4 and 5) were

consistently detected across both replicates and were deemed stable QTL (Fig 3, Table

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 3). Stable QTL were identified on chromosomes 3A (Rpsphq1), 3D (Rpsph2) and 5B/7B
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(Rpsphq5) and two QTL were identified on chromosome 4A (Rpsphq3 and Rpsphq4)

(Table 3, Fig. 3 and Supplementary Fig. 2). All stable QTL were contributed by the

resistant AvR parent (Table 3).

Discussion

The ability to conduct genetic studies of nonhost resistance in cereal – Puccinia

pathosystems is often hampered by a lack of fully susceptible plant genotypes. Previous

genetic studies on the resistance to heterologous rust pathogens in wheat, including

barley crown rust (P. coronata var. hordei- Pch) and rye stem rust (Pgs), were either

dependent on rare susceptible genotypes or the development of susceptible

experimental lines (Sanghi 1968; Jin and Steffenson 1993; Nui et al., 2014). These

previous studies and the current study suggest that wheat is an intermediate or near-

nonhost to Pch, Pgs and Psph respectively. This conclusion was based on the observed

susceptibility of only a few wheat landraces and the single Australian wheat cultivar

Teal to Psph. Niks and Marcel (2009) argue that assuming that cereal rust pathogens co-

evolve with particular grass host species, it is not unreasonable to expect that some

grass species maybe in the process of either losing or acquiring host status to specific

Puccinia species and therefore take an intermediate position. Atienza et al., (2004)

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 determined that rare susceptibility can occur in barley to most heterologous rust
Accepted Article
pathogens, it appears wheat may also be an intermediate host to some cereal rust

pathogens but not others.

Nonhost interactions are reported to involve multiple layers of different

resistance mechanisms (Stam et al., 2014; Niks and Marcel 2009). The higher frequency

of landraces with intermediate or susceptible reactions relative to the widespread

immunity observed in nearly all cultivated wheats to Psph was consistent with the

results of Atienza et al., (2004), who assessed the response of 110 diverse barley

accessions to more than 10 different heterologous rust pathogens. Presumably, the

widespread immunity observed on cultivated wheats relative to the landraces in

response to Psph may be due to the accumulation of additional QTL through

preferential selection of partial resistance to adapted rust pathogens or the genetic

diversity among the cultivars tested was low.

The observed necrotic and chlorotic ITs on the wheat landraces assessed may

suggest the involvement of post-haustorial resistance mechanisms possibly suggesting a

gene for gene mode of inheritance, however histological comparisons are required to

confirm such a hypothesis. The phenotypic assessment of different wheat accessions

including the wheat stripe rust differential set in the present study indicated that either

many wheat stripe rust resistance genes may also be effective to Psph and/or that there

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 are specific common genes in wheat that confer resistance to Psph but not Pst. The stem
Accepted Article
rust resistance gene Sr18 was shown to be present in 95% of wheats and confers

resistance to Pgs but not Pgt (Sanghi 1968). Numerous studies in barley have compared

the overlapping specificities of resistance QTL in response to heterologous rust

pathogens and partial resistance to the adapted barley leaf rust pathogen P. hordei (Niks

1983; Marcel et al., 2007; Jafary et al., 2008; Yeo et al., 2014; Niks et al., 2015). A recent

study also determined that not only QTL but common resistance genes such as the rye

stem rust resistance locus Rpg5 in barley conferred resistance to multiple formae speciales

of P. graminis (Dracatos et al., 2015).

In wheat, the complementary resistance genes Yr73 (3DL) and Yr74 (5BL) in AvR

confer resistance to the original Pst pathotype group introduced into Australia from

Europe (104 E137 A-) and all its mutational derivatives (O’Brien et al., 1979; Wellings et

al., 1988; Dracatos et al., 2016). In the present study AvR had a similar yet lower IT in

response to Psph compared with avirulent Pst pathotypes, therefore we hypothesized

that complementary genes Yr73 and Yr74 may also confer near-nonhost resistance to

Psph. To test this hypothesis we used the same DH mapping population (Teal/AvR) as

Dracatos et al., (2016) due to the observed susceptibility of Teal to Psph to map

resistance to Psph in AvR. Without exception, lines carrying the AvR complementary

resistance were resistant to Psph, suggesting that either the same or very closely linked

genes conferred resistance to both Pst and Psph. The presence of DH lines, resistant to

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 Psph and susceptible to Pst pts. 104 E137 A- or 108 E141 A-, suggests the presence of
Accepted Article
additional resistance gene loci in AvR that confer resistance to Psph and not Pst.

The association of complementary resistance genes Yr73 and Yr74 with resistance

to Psph in AvR was also strongly supported by the genetic mapping results observed in

this study. Previous genetic mapping in the same DH population as used in this study

determined that resistance was simply inherited and Yr73 and Yr74 mapped to

chromosomes 3DL and 5BL (Dracatos et al., 2016). Here we determined that resistance

to Psph in wheat is more complex and polygenically inherited. According to Nui et al.,

(2014), the resistance in wheat cv. Chris to Pch was simply inherited and due to a single

dominant gene on chromosome 5D. All forms of mapping analysis used in this study

identified stable resistance QTL to Psph on chromosomes 3DL and 5BL in the same

regions as Yr73 and Yr74 respectively, in addition to two QTLs on chromosome 4AS

and 3A. Previously designated wheat stripe resistance genes have been mapped on

chromosomes 4AL (Yr51 and Yr60) (Randhawa et al., 2014; Herrera-Foessel et al., 2015),

however the QTL identified here was on 4AS suggesting that the above genes are not

involved in resistance to Psph. To date no Yr genes have been designated from

chromosome 3A suggesting this QTL may represent a gene specific to Psph.

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 Phenotypic assessment of selected T/A DH lines with the barley stripe rust
Accepted Article
pathogen, Psh determined that lines carrying Yr73 and Yr74 in combination also

displayed consistently lower ITs than the DH lines and Teal that lacked both

complementary genes. Further genetic studies are required based on intercrossing lines

carrying either Yr73 and/or Yr74 with Psh-susceptible wheat accessions such as PI-

478214 reported by Pahalawatta and Chen (2005). If indeed Yr73 and Yr74 confer

resistance to multiple formae speciales of P. striiformis it may be hypothesized that non-

adapted P. striiformis isolates such as Psph and Psh, share common avirulence genes

with Pst. Both Johnson and Lovell (1994) and Pahalawatta and Chen (2005) conducted

genetic studies on the genetic basis of resistance in wheat to the barley stripe rust

pathogen. Pahalawatta and Chen (2005) identified a single dominant gene was effective

to multiple races of Psh and was closely linked to the resistance gene for Pst race PST-21

Yr21 on chromsosome 1B. A QTL for resistance to Psph was identified in this study on

chromosome 1B, however further studies are required to determine the relationship of

the 1B QTL with the resistance in Lemnhi or Yr21. Nevertheless, both single gene and

gene combination lines in the wheat T/A DH mapping population identified in the

present study, along with barley genotypes carrying single genes for resistance to Psph

identified in previous genetic studies, will be used to monitor possible changes

virulence in Psph populations.

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 Acknowledgments
Accepted Article
The authors acknowledge the Grains Research Development Corporation, The

Australian Research Council and the Endeavour Scholarships program for funding this

research. The authors also thank Professor Robert McIntosh for critical reading of this

manuscript and Associate Professor Colin Wellings for the provision of the

Teal/AvocetR doubled haploid population. The authors also thank Dr Davinder Singh

for provision of wheat seed for testing with Psph. The authors also thank Mrs Amelia

Hubbard from the National Institute of Agricultural Botany, Cambridge UK for the

provision of Psh isolate for use in experiments. The authors acknowledge Diversity

Array Technologies (DArT-Canberra) for the genotypic analysis, genetic map

construction and marker trait analysis performed for this study on a fee-for-service

basis.

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Figure Legends

Figure 1

Seedling leaves (L to R): A. 11 USDA-derived wheat landraces (Cltr-7611, PI-554450, PI-

573183, PI-321947, PI-165144, PI-565242, Cltr-7599, PI-117754, PI-192569, PI-321947, PI-

349517), Teal and the barley cultivar Maritime and B. Teal, AvocetR, Teal/AvR doubled

haploid lines T/A2 (susceptible), T/A63 (Yr73), T/A72 (Yr74), T/A13 (Yr73 and Yr74),

Chinese 166 (Yr1) and Avocet S (Yr73) and infected with P. striiformis f. sp. pseudo-hordei

(BGYR isolate 981549) 18 days after inoculation.

Figure 2

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 Frequency distribution for infection response data for A. percentage of wheat accessions
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tested including 245 wheat landraces (black) and 393 cultivated wheats (grey) (total

number = 638 accessions) and B. the number of Teal/AvocetR DH lines in response to P.

striiformis f. sp. pseudo-hordei (BGYR isolate 981549) (total number of DH lines

represented = 86). Black and grey bars represent the first and second experimental

replicates respectively.

Figure 3

Selected linkage groups of the genetic map for the Teal/AvocetR wheat DH population

(Dracatos et al., 2016) showing the stable QTLs identified in both phenotypic replicates

(green and blue corresponding to replicates 1 and 2 respectively). The name on QTL

bars has two components: the provisional name of the gene at the QTL and the LOD

value recorded for the QTL. Each QTL bar shows the LOD-1 support interval, the

exceeding lines the LOD-2 support interval of each QTL, based on MTM CIM results.

The length of the coloured solid bars indicates the LOD-1 confidence intervals (with

their corresponding peak markers in black bold) while the QTL lines are extended to

the LOD-2 confidence intervals.

Supplementary Figure 1

Estimated recombination fractions for all pairs of markers (on selected chromosomes),

along with LOD scores for the test of r = 1/2. (The recombination fractions are in the

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 upper left triangle; LOD scores are in the lower right triangle.) Yellow indicates a large
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LOD score or a small recombination fraction.

Supplementary Figure 2

QTL LOD trace file of both replicates (dotted line=replicate 1 and solid line = replicate 2)

of the Composite Interval Mapping results for resistance to Puccinia striiformis f. sp.

pseudo-hordei. For both replicates a LOD threshold of 2.5 was used to determine the

presence of a QTL.

Supplementary Table 1

Details of diverse accessions (landraces and cultivated bread wheats) phenotypically

assessed at the seedling stage with Puccinia striiformis f. sp. pseudo-hordei.

Supplementary Table 2

Comparative infection type, quantitative scores and marker data for the wheat Teal x

AvocetR doubled haploid mapping population in response to the adapted (P. striiformis

f. sp. tritici) and heterologous (P. striiformis f. sp. pseudo-hordei) stripe rust pathogens.

Supplementary Table 3

Teal x AvocetR genetic map including annotations to consensus wheat physical and

genetic maps.

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 Table 1 Response of wheat lines and a barley selection to two Australian pathotypes of Puccinia
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striiformis f. sp. tritici (Pst) that differ in their virulence on Avocet R, and to P. striiformis f. sp.
pseudo-hordei (Psph) isolate 981549

Differential genotype Gene Pst 108 E141 A+ Pst 108 E141 A− Psph 981549
Chinese 166 Yr1 0; 0; 2CN
Lee Yr7 ;CN ;CN ;N
Heines Kolben Yr6 3 3 ;0;
Vilmorin 27 Yr3 3− 3− 0; −
Moro Yr10 0; 0; 0; −
Strubes Dickkoph Yr2 2C/3 2C/3 0;/;
Suwon 92/Omar Yr4 3 3 0;
Clement Yr9 0; 0; 0;/; −
Triticum Spelta Yr5 0; 0; 0; −
Hybrid 46 Yr4 3=C 3=C 0;
Reichersberg 42 Yr7 ;CN ;CN 0; −
Heines Peko Yr6 3 3 0; −
Nord Desprez Yr3, Yr4 3+ 3+ 0; −
Compair Yr8 0; 0; 0; −
Carstens V Yr32 ;CN ;CN 0; −
Spaldings Prolific Yrsp ;CN ;CN 0; −
Heines VII Yr2 3−C 3 −C 0; −
Avocet R YrA (Yr73, Yr74) 33+ 12CN ;C
Kalyansona Yr2 3+C 3 +C 0; −
Trident Yr17 ;C+N ;C+N 0; −
Yr15 NILa Yr15 0; 0; 0;
Hugenoot Yr25 3−N 3 −N 0; −
Selkirk Yr27 ;NN ;NN 0;/;N
Yr9 NILa Yr9 ;N ;N 0; −
Avocet S Yr73 3+ 3+ 0;
EGA Gregory Yr33 2+N 2 +N 0; −
Ellison Yr17 0; 0; 0; −
Tobruk YrT ;N ;N 0;-
Breakwell YrJ 0;/;C 0;/;C 0;
Yr17 NILa Yr17 ;C1 ;C1 0; −
Morocco — 33+ 33+ ;
Clipper/Saharab — CC/;C CC/;C 33+
0 = no visible symptoms, ; = necrotic flecks, ;N = necrotic areas without sporulation, 1 = necrotic and
chlorotic areas with restricted sporulation, 2 = moderate sporulation with necrosis and chlorosis, 3 =
sporulation with chlorosis, 4 = abundant sporulation without chlorosis. Variations of the ITs were
indicated by use of − (less than average for the class), + (more than average for the class), C (chlorosis)
and/or N (necrosis).

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 a
NIL refers to near isogenic lines based on Avocet S.
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b
Susceptible barley selection from the Clipper × Sahara doubled haploid mapping population.

Table 2 Phenotypic infection type data for parental and selected doubled haploid lines from the wheat
Teal × Avocet R mapping population in response to two Puccinia striiformis f. sp. tritici (Pst) pathotypes
differing in virulence to YrA and single pustule-derived isolates of P. striiformis ff. sp., P. striiformis f.
sp. pseudo-hordei (Psph) and P. striiformis f. sp. hordei (Psh)

Resistance
Wheat line gene(s) Pst 108 E141 A− Pst 108 E141 A+ Psph 981549 Psh 83.39

Teal None 4 4 3++ ;CN

Avocet R Yr73, Yr74 ;C1− to 2CN 4 ;CN ;C

T/A112 Yr73, Yr74 3C 4 2−CN ;N

T/A113 No datab 4 4 2++C ;NN

T/A115 Yr73, Yr74 2C 4 3+ ;CN

T/A13 Yr73, Yr74 2+C 4 2+NN ;CN

T/A25 Yr73, Yr74 2C 4 1N ;;CN

T/A26 Yr73, Yr74 ;CN 4 ;NN ;NN

T/A3 Yr73, Yr74 12−C 4 ;CN ;CN

T/A45 No datab 4 4 CN1− 12+NN

T/A48 Yr73, Yr74 ; 4 0; 0;

T/A49 Yr74 4 4 1NN 1NN

T/A50 Yr73 4 4 ;CN 1NN

T/A51 Yr73, Yr74 ;C 4 ;CN ;CN

T/A76 Yr73, Yr74 ;C 4 ;CN ;;=C

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 T/A77 Yr73, Yr74 ;;C 4 1CN ;;C
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Morocco None 3+ 3+ 2+, ;C2+ ;C

Chinese 166 Yr1 ; ; 2CN N1−

0 = no visible symptoms, ; = necrotic flecks, ;N = necrotic areas without sporulation, 1 = necrotic and
chlorotic areas with restricted sporulation, 2 = moderate sporulation with necrosis and chlorosis, 3 =
sporulation with chlorosis, 4 = abundant sporulation without chlorosis. Variations of the ITs are indicated
by use of − (less than average for the class), + (more than average for the class), C (chlorosis) and/or N
(necrosis).

a
The presence of resistance genes was postulated for the presence of the closely linked DArT-Seq markers
to Yr73 and Yr74 (Dracatos et al., 2015).

b
No data refers to lines of the doubled haploid population that were phenotyped but not genotyped.

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ccepted Articl Table 3 Significant stable quantitative trait loci (QTL), derived from two experimental replicates, conferring resistance to barley grass
stripe rust (Puccinia striiformis f. sp. pseudo-hordei) in the wheat Teal × Avocet R doubled haploid mapping population

Linkage Position Max

QTL Replicate group (cM) Peak marker LODa SMR Pr (F)b Ac Donor
Rpsphq1 1 3A_1 15.11 1090388|F|0-61:A>G-61:A>G 6.01 0.000**** 0.44 Avocet R
2 3A_1 15.11 1090388|F|0-61:A>G-61:A>G 4.05 0.000*** 0.37 Avocet R
Rpsphq2 1 3D_3 40.5 1160719|F|0 3.71 0.000**** −0.41 Avocet R
2 3D_3 40.48 1160719|F|0 3.49 0.000**** −0.36 Avocet R
Rpsphq3 1 4A_2 0 1269095|F|0 3.89 0.000**** 0.45 Avocet R
2 4A_2 0 1269095|F|0 4.63 0.000**** 0.48 Avocet R
Rpsphq4 1 4A_2 43.9 1138925|F|0 3.8 0.000**** 0.46 Avocet R
2 4A_2 43.9 1138925|F|0 4.41 0.000**** 0.49 Avocet R
Rpsphq5 1 5B/7B 83.8 100002679|F|0 3.11 0.000*** 0.40 Avocet R
2 5B/7B 83.8 100002679|F|0 4.13 0.000**** 0.46 Avocet R
a
Logarithm of odds.

b
Single marker regression probability.

c
Additive effect of substituting alternative alleles at marker locus.

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