Mapping and diagnostic marker development for Soil-bornecereal mosaic virus resistance in bread wheat

Dragan Perovic Æ Jutta Forster Æ Pierre Devaux Æ Djabbar Hariri Æ Morgane Guilleroux ÆKostya Kanyuka Æ Rebecca Lyons Æ Jens Weyen Æ David Feuerhelm Æ Ute Kastirr ÆPierre Sourdille Æ Marion Roder Æ Frank Ordon

Received: 18 June 2008 / Accepted: 22 January 2009 / Published online: 12 February 2009

� Springer Science+Business Media B.V. 2009

Abstract Monogenically-inherited resistance to

Soil-borne cereal mosaic virus (SBCMV) in hexa-

ploid bread wheat cultivars ‘Tremie’ and ‘Claire’ was

mapped on chromosome 5D. The two closest flanking

markers identified in the Claire-derived mapping

population, Xgwm469-5D and E37M49, are linked to

the resistance locus at distances of 1 and 9 cm,

respectively. Xgwm469-5D co-segregated with the

SBCMV resistance in the Tremie-derived population

and with the recently identified Sbm1 locus in the cv.

Cadenza. This suggested that Tremie and Claire carry

a resistance gene allelic to Sbm1, or one closely

linked to it. The diagnostic value of Xgwm469-5D

was assessed using a collection of SBCMV resistant

and susceptible cultivars. Importantly, all susceptible

genotypes carried a null allele of Xgwm469-5D,

D. Perovic � F. Ordon (&)

Julius Kuehn-Institute, Federal Research Centre for

Cultivated Plants, Institute for Resistance Research and

Stress Tolerance, Erwin-Baur-Str. 27, 06484 Quedlinburg,

Germany

e-mail: frank.ordon@jki.bund.de

D. Perovic

e-mail: dragan.perovic@jki.bund.de

J. Forster � J. Weyen

Saaten-Union Resistenzlabor GmbH, Hovedisser Str. 92,

33818 Leopoldshohe, Germany

P. Devaux

Florimond Desprez, 3, Rue Florimond Desprez, 59242

Cappelle en Pevele, France

D. Hariri � M. Guilleroux

INRA, BIOGER, Route de Saint Cyr, 78026 Versailles

Cedex, France

K. Kanyuka � R. Lyons

Department of Plant Pathology and Microbiology, Centre

for Sustainable Pest and Disease Management,

Rothamsted Research, Harpenden, AL5 2JQ

Hertfordshire, UK

D. Feuerhelm

Elsoms Seeds Ltd, Pinchbeck Road, PE11 1QG Spalding,

UK

U. Kastirr

Institute for Epidemiology and Pathogen Diagnostics,

Julius Kuehn-Institute, Federal Research Centre for

Cultivated Plants, Erwin-Baur-Str. 27, 06484

Quedlinburg, Germany

P. Sourdille

UMR 1095 Amelioration et Sante des Plantes, INRA,

Domaine de Crouelle 234, Avenue du Brezet, Clermont

Ferrand, France

M. Roder

Leibniz Institute of Plant Genetics and Crop Plant

Research (IPK), Corrensstr. 3, 06466 Gatersleben,

Germany

123

Mol Breeding (2009) 23:641–653

DOI 10.1007/s11032-009-9262-2

whereas resistant genotypes presumably related to

either Claire and Tremie or Cadenza revealed a 152

or 154 bp allele of Xgwm469-5D, respectively.

Therefore, Xgwm469-5D is well suited for marker

assisted selection for SBCMV resistance.

Keywords Soil-borne cereal mosaic virus

(SBCMV) � Winter wheat (Triticum aestivum) �Resistance � Sbm1 � Marker assisted selection (MAS)

Introduction

Mosaic disease, caused by Soil-borne cereal mosaic

virus (SBCMV), which belongs to the genus Furovi-

rus, is a serious constraint to winter wheat production

in Europe, North America and Asia (Clover et al.

2001). SBCMV is transmitted by the plasmodiophorid

Polymyxa graminis, a eukaryotic soil-borne micro-

organism that has been detected down to a soil depth

of 60 cm and colonises roots of Gramineae plants

(Rao and Brakke 1969). Following transmission by

P. graminis, SBCMV is translocated into the upper

parts of susceptible plants causing stunting and

mosaic symptoms on leaves that are most prominent

in early spring. Winter wheat plants infected in

autumn are particularly sensitive to frost damage

resulting in increased winter killing or reduced vigour

during spring. Chemical control except soil fumiga-

tion, which is unacceptable for economical and

ecological reasons, is ineffective against P. graminis.

Furthermore, as virus-containing resting spores of

P. graminis are distributed by wind, water and

machinery and can survive in the soil for decades

(Brakke and Langenberg 1988), crop rotation is not an

effective option for disease control either. Therefore,

the only possibility of controlling this disease on

infested fields is the growing of resistant cultivars.

Historically, Soil-borne wheat mosaic virus

(SBWMV) was first recognized as a serious winter

wheat disease in Indiana and Illinois in 1919 (Mc-

Kinney 1923) and has subsequently been detected in

other winter wheat growing areas around the globe

(Budge and Henry 2002; Proeseler and Stanarius

1983; Saito et al. 1964). In Europe, however, the most

common causal agent of mosaic disease of wheat

(Triticum aestivum), durum wheat (Triticum durum),

rye (Secale cereale) and triticale is SBCMV, a virus

species related to SBWMV and until recently

considered as a European strain of SBWMV. Com-

plete sequencing of SBWMV and SBCMV genomes

revealed that they share only approximately 70%

nucleotide identity, and therefore should be classified

as separate species (Koenig and Huth 2000; Torrance

and Koenig 2004). In addition, it has been shown that

these two viruses can be distinguished biologically by

infection phenotype on the indicator plant Nicotiana

benthamiana. Whereas SBCMV spreads systemically

and causes leaf chlorosis and subsequent plant

death, SBWMV is restricted to the inoculated leaves

(Kastirr et al. 2006). More recently, it has also been

shown that SBWMV and SBCMV can be distin-

guished by their infection phenotype on some barley

genotypes (Lyons et al. 2008).

SBCMV-resistant wheat cultivars exhibit the

so-called ‘translocation resistance’ (Hariri et al.

1987). In such cultivars, virus accumulates to a

certain degree in the roots, but its movement from the

roots to the stem and leaves is blocked or delayed

such that the viral coat protein antigen is undetect-

able in above ground tissues. Resistant cultivars have

been known in France for many years, but until now

no comprehensive information on the genetic control

of resistance in these cultivars have been available.

Very recently, a locus for resistance to SBCMV,

designated as Sbm1, has been mapped to the long

arm of chromosome 5D in the UK wheat cv. Cadenza

(Bass et al. 2006). However, it appears that Cadenza

shares no common ancestry, regarding donors of

resistance, with SBCMV-resistant cultivars com-

monly grown in France (e.g., Tremie, Claire and

Moulin) (Bayles and Napier 2002). Therefore, the

present study aimed at obtaining detailed information

on the genetics of SBCMV resistance in elite

European wheat cultivars that are unrelated to cv.

Cadenza, and developing molecular markers which

can be employed for efficient marker assisted selec-

tion (MAS) in breeding for SBCMV resistance.

Development of diagnostic markers for rapid identi-

fication of SBCMV resistant genotypes is of special

importance in wheat breeding as field based pheno-

typic selection is expensive, laborious and time

consuming.

642 Mol Breeding (2009) 23:641–653

123

Materials and methods

Plant materials

Doubled haploid (DH) wheat populations segregating

for resistance to SBCMV were derived from crosses

between the resistant cv. Tremie and the susceptible

cultivars Texel, Aztek and Soissons (in total 64 DH

lines), and between the resistant cv. Claire and the

susceptible cv. Savannah (126 DH lines) using inter-

generic hybridisation with maize (Devaux and

Pickering 2005; Table 1). In addition, the population

of 204 DH individuals, derived from a cross between

susceptible cv. Avalon and resistant cv. Cadenza

(Kanyuka et al. 2004) was used for linkage analy-

sis between Sbm1 and the microsatellite marker

Xgwm469. For chromosomal and deletion bin assign-

ment of molecular markers, a set of 21 nulli-tetrasomic

chromosome substitution lines (Sears 1954, 1966) and

one deletion line 5DL5 of the wheat cv. Chinese

Spring (Endo and Gill 1996; Werner et al. 1992) was

used. The diagnostic value of the markers linked to

SBCMV resistance was assessed using a genetically

diverse collection of 99 wheat cultivars that were

tested for resistance to SBCMV (Table 2). Seeds of

these genotypes were obtained primarily from the

National Small Grains Collection, USDA-ARS, Aber-

deen, Idaho, USA.

SBCMV resistance tests

In order to assess resistance of segregating DH

populations, visual scoring and Double Antibody

Sandwich Enzyme-Linked Immunosorbent Assay

(DAS-ELISA) were carried out on plants grown in

highly and uniformly infested soils. DH populations

were evaluated during two growing seasons (2004/

2005 and 2005/2006) at two different locations in

France (Vatan) and Germany (Walternienburg). Each

DH line and parental cultivar was grown as a double-

row (30 plants per row) with two replications at each

location. The method for visual disease scoring was

as follows: all lines were scored on a scale of 0–9 for

appearance of yellow mosaic on leaves. A score of

‘0’ indicated no plants displaying yellow mosaic are

present, and a score of ‘9’ indicated 80–100% of

plants with yellow mosaic symptoms are observed.

DAS-ELISA was performed according to Clark and

Adams (1977). For each DH line ten plants individ-

ually or in two bulks of five plants were ground in a

0.1 M citrate buffer (pH 7.2) containing 0.5 M urea

and centrifuged at 10,000 g for 3 min. The superna-

tants were collected and 100 ll aliquots were added

to each well, which previously had been coated with

SBCMV IgG (1 lg ml-1). After incubation overnight

at 4�C, plates were washed three times and the

alkaline phosphatase-conjugated IgG were added.

After incubation for 4 h at 37�C and a further

washing step, p-nitrophenyl phosphate substrate was

added and extinction was measured at 405 nm.

Samples were considered to be positive when the

E450 value was more than three times that of the

healthy control. In disease scoring three distinct

phenotypic classes were observed as follows: 1)

resistant (with ELISA values B 0.3 and visual symp-

toms scores of ‘0’–‘3’), 2) moderately resistant (with

ELISA values 0.3–0.5 and visual symptoms scores of

‘3’–‘5’), and susceptible (with ELISA values C 0.5

and visual symptoms scores of ‘5’–‘9’).

An additional collection of 99 wheat genotypes of

European and ‘foreign’ origin with known or putative

resistance to furoviruses was assembled for SBCMV

resistance screening and SSR analyses. Plants were

grown in SBCMV-infested soils collected from an

Table 1 Pedigree and segregation ratios of DH-populations used for mapping SBCMV resistance in wheat

Cross Number of DH

lines in progeny

Number of

resistant DHs

Number of

susceptible DHs

v2(1r:1s) P df

Tremie 9 Aztek 17 – – – – –

Tremie 9 Texel 17 – – – – –

Tremie 9 Soissons 30 – – – – –

Tremie-derived population (combined) 64 27 37 1.56 0.211 1

Claire 9 Savannah 126 69 57 1.14 0.285 1

v2, chi-square test; P, probability; df, degrees of freedom

Mol Breeding (2009) 23:641–653 643

123

Table 2 Winter wheat genotypes screened with SSR WMS469 and their reactions to SBCMV

No. Accession identifiera Acsession name Reaction to SBCMV Xgwm469-5DL allele Xwmc765

1 PI 81793 Akagawa Ako Ichigo R 154 179

2 Alixan R 154 nt

3 PI 340666 Alpha R 152 195

4 Altigo R 154 nt

5 Arsene R 152 nt

6 Autan R 152 nt

7 Avalon S – 199

8 Axima R 152 nt

9 Axona S – 191

10 Aztek S – 193

11 Badger S – 221

12 Biscay S – 193

13 Buster S – 195

14 Cadenza R 154 209

15 Charger R 152 199

16 Chatsworth S – 179

17 Chinese Spring S – 179

18 PI 338915 Ciano F67 S – 191

19 Claire R 152 193

20 Cltr 17882 Line B S – 199

21 Cltr 17886 Line G R 152 199

22 CItr 11673 Comanche R 154 209

23 Consort S – 195

24 Corvus S – 199

25 Deben S – 195

26 Domino S – 191

27 PI 414565 Dong Fang Hong 3 MR – 177

28 PI 361730 Dronning Wilhelmine II S – 189

29 PI 591964 Empraba 16b S – 225

30 Ephoros S – 189

31 PI 262231 Etoile de Choisy MR 154 187

32 PI 476850 F�S. 401 MR 152 167

33 Flame R 152 199

34 Hadm10437-1 S – 193

35 PI 340707 Heines C73 R 154 209

36 Hereward R 152 199

37 CItr 17264 Homestead MR 152 195

38 PI 405702 Hong Liang 5b S – 191

39 PI 495015 IL 77-4259 MR 154 179

40 PI 593688 Jagger R 154 179

41 Kharchia Local S – 167

42 PI 351622 Klein Rendidor MR 152 191

43 PI 566671 KS92WGRC22b S – 179

44 PI 566670 KS92WRGC21b MR – 179

644 Mol Breeding (2009) 23:641–653

123

Table 2 continued

No. Accession identifiera Acsession name Reaction to SBCMV Xgwm469-5DL allele Xwmc765

45 Malacca S – 195

46 Mirage R 152 nt

47 Moulin R 152 199

48 CItr 17715 Newton R 154 179

49 CItr 15929 Oasis MR 154 209

50 PI 372428 Odesskaja 51 R 154 209

51 Okostar R 152 211

52 CItr 8220 Oro R 154 213

53 Paladain R 152 nt

54 PI 316438 Odesskaja 16 S – 213

55 PI 46799 CI 6225 S – 195

56 PI 499691 KS85WGRC01 R 152 181

57 PI 527480 Karl S – 195

58 PI 532914 2555 R 152 181

59 PI 552816 Howell R 152 223

60 PI 552985 TX73A2798 S – 181

61 PI 564245 Karl 92 R 152 nt

62 PI 574488 Ike S – 181

63 PI 587171 Hazen R 152 171

64 PI 592444 KS92P0263-137 R 152 189

65 PI 592750 Glory R 152 189

66 PI 592760 Jaypee R 152 169

67 PI 608672 Goldfield R 152 169

68 PI 631493 Ok101 S – 181

69 Piko S – 193

70 Pitic 62 S – nt

71 PI 591702 Plainsman V MR 154 179

72 Plutos R 154 nt

73 PI 351886 Porvenir R 154 195

74 CItr 3392 Red Chief MR 154 179

75 Rialto S – 217

76 Riband S – 195

77 Robigus S – nt

78 Savannah S – 195

79 Scorpion 25 S – 219

80 PI 64111 Semka MR 154 209

81 Senat Nt 152 193

82 Shamrock S – 219

83 CItr 14157 Shawnee R 154 215

84 CItr 6163 Shepherd R 154 183

85 Soissons S – 193

86 Solstice S – 193

87 Sonora 64 R 154 181

88 Spark Nt 152 199

Mol Breeding (2009) 23:641–653 645

123

infested site in Wiltshire, UK or Kent, UK mixed

with two parts soil and 3.0–3.5 g/l of the controlled

release fertiliser Osmocote Plus (Scotts Europe) in a

controlled temperature glasshouse as described in

Bass et al. (2006). Two separate tests were undertaken

during 2005, with sowings in March and June. For each

test, eight plants were sown per cultivar. At 8 and

10 weeks post planting, the youngest fully expanded

leaf from each plant was collected and leaves from

each pot were combined for ELISA. Sample prepara-

tion and virus antigen detection by indirect F(ab0)2

ELISA was performed as described in Bass et al.

(2006). Two replicates of each sample, including an

uninfected and infected control, were tested per assay.

Samples with ELISA values greater than three times

the uninfected control were considered positive.

Genotypes were scored for SBCMV resistance as

follows: R (resistant): similar response to the resistant

UK cv. Cadenza: virus antigen detected in 0–25%

samples; MR (moderately resistant): virus antigen

detected in 25–50% of samples; S (susceptible), similar

response to the susceptible UK cultivar Avalon: virus

antigen detected in 50–100% of samples.

DNA extraction and molecular mapping

Genomic DNA was isolated by the CTAB method

according to Graner et al. (1991) with an additional

extraction with chloroform:isoamyl alcohol (24:1)

before DNA precipitation with alcohol was performed.

Phenotypic data for the SBCMV resistance

obtained by visual scoring of genotypes grown in

SBCMV-infested fields and DAS-ELISA were used

for composing resistant and susceptible DNA bulks

according to Michelmore et al. (1991). DNA from

five to eleven resistant and susceptible DH lines,

respectively, were combined per bulk for each DH

population separately and used in SSR and AFLP

analyses. Resistant and susceptible bulks were

screened with 256 EcoRI ? 3/MseI ? 3 AFLP pri-

mer combinations and products were resolved either

on an automated laser fluorescence A. L. F. express

sequencer (Amersham Biosciences) and analyzed

using the computer program Fragment Analyzer

v.1.02 (Amersham Biosciences), or on a CEQTM

8000 Genetic Analysis System (Beckman). Digestion

and amplification of AFLPs were performed accord-

ing to the ‘AFLP Core Kit manual’ (Invitrogen).

In addition, a total of 162 genomic and EST-

derived microsatellite markers located on 21 wheat

chromosomes were amplified as previously described

(Roder et al. 1998; Somers et al. 2004; Zhang et al.

2005). An improved PCR profile for Xgwm469 was

obtained by lowering the annealing temperature to

50�C, and by adding an extra 7-bp PIGtail sequence

(Brownstein et al. 1996) to the 50-end of the reverse

primer while other PCR steps remained the same as

described by Roder et al. (1998). Due to this extra

tail, the fragment size of 152 and 154-bp of

Xgwm469-5D increased to 159 or 161-bp,

Table 2 continued

No. Accession identifiera Acsession name Reaction to SBCMV Xgwm469-5DL allele Xwmc765

89 PI 351465 Teverson R 154 187

90 Texel S – 193

91 Tonic R 154 209

92 Tremie R 152 199

93 PI 11610 Turkey MR 152 191

94 PI 355947 Ul’ianovka S – 179

95 Vivant S – 193

96 Warlock 24 S – 219

97 Wasp S – 193

98 PI 477288 Wrangler R 154 181

99 Xi-19 S – 219

a Accession identifier in National Small Grains Collection, USAb Cultivars that are thought to be resistant to SBWMV in previous studies but shown to be susceptible to SBCMV in this study

‘–’, Null allele; nt, Not tested

646 Mol Breeding (2009) 23:641–653

123

respectively. Amplifications were carried out in a

AB9700 Thermal Cycler (Applied Biosystems) or a

DNA Engine PTC-200 Thermal Cycler (MJ

Research) using fluorescently labelled (Cy5 or

DY-681) forward and unlabeled reverse microsatel-

lite primers. Products were resolved either on the

A. L. F. ExpressTM automated laser fluorescence

sequencer (Amersham Biosciences), on the CEQTM

8000 Genetic Analysis System (Beckman), or on the

NEN Model 4300 DNA Analyzer (Li-Cor) as per the

manufacturer’s instructions.

Computational linkage analysis was carried out

using Joinmap v.3.0 (Stam 1993) software.

Results

Disease scoring

Visual evaluation and DAS ELISA-based disease

scoring of 190 DH lines during two field seasons

at two geographical locations (i.e., France and

Germany) yielded a uniform and consistent set of

data for all progeny plants in both mapping popula-

tions. Phenotypic and ELISA data were highly and

significantly correlated, i.e., 0.95*** and 0.89***,

respectively, in the different DH-populations. In

general, two distinct phenotypic classes were

observed, since only a small proportion of DH lines,

five and nine DH lines in Tremie- and Claire-derived

populations, respectively, had an intermediate reac-

tion with visual symptom scores between ‘3’ and ‘5’

and ELISA values 0.3–0.5 Therefore, both resistance

classes (resistant and moderately resistant) were

scored as resistant. Observed segregation of pheno-

types in DH populations were 27R:37S and 69R:57S

for Tremie- and Claire-derived populations, respec-

tively (Table 1). The segregation ratios (v2 = 1.56;

P = 0.211 and v2 = 1.14; P = 0.285; 1df) fit to a

1R:1S segregation indicating that resistance is con-

trolled by one major locus, while occurrence of 14

lines with moderate resistance out of 190 examined

may indicate the presence of another minor gene.

This result is in correspondence with previous

findings for inheritance of SBCMV resistance in

wheat (Bass et al. 2006; Kanyuka et al. 2004; Perovic

et al. 2005).

A disease response score of resistance (R), mod-

erate resistance (MR) or susceptibility (S) was

applied to the 99 wheat accessions inoculated with

SBCMV-infested soils from Wiltshire or Kent, UK in

a controlled temperature glasshouse (Table 2). Most

of the accessions were clearly identified as either R or

S confirming the previous published or unpublished

observations by either us or other research groups.

Several very old wheat accessions (e.g., Turkey) that

are thought to be resistant to soil-borne viral mosaic

disease, however, were classified as MR. Also, quite

unexpectedly, some wheat accessions (e.g., Embrapa

16 and KS92WGRC21) that have been previously

described as resistant to soil-borne viral mosaic

disease in North and/or South America (Barbosa

et al. 2001; Cox et al. 1994) were fully susceptible to

SBCMV in our tests.

Genetic mapping and marker saturation of the

SBCMV resistance locus

Due to the monogenic inheritance of SBCMV

resistance and the four populations analysed, eight

distinct DNA bulks for screening with microsatellite

and AFLP markers were constructed. A total of 162

EST-derived and genomic microsatellite markers and

256 EcoRI ? 3/MseI ? 3 AFLP primer combina-

tions were surveyed to identify polymorphisms

between the resistant and the susceptible DNA bulks

and the six parental lines of the mapping populations.

Initially, a set of 130 genomic GWM (Gatersleben

wheat microsatellites) markers distributed over the 21

chromosomes of the wheat genome were surveyed for

polymorphism. Screening revealed 78 monomorphic

markers, while 52 were polymorphic in at least one of

the four parental combinations used. The initial

screening of DNA bulks detected Xgwm469 as

polymorphic on parents as well as on bulks of the

Tremie-derived population, and Xgwm272 as poly-

morphic on parents as well as on bulks of the Claire-

derived population. Genotyping of the three small

Tremie-derived populations did not detect linkage of

the most prominent Xgwm469 fragment of 170-bp

and the SBCMV resistance locus, while careful

scoring of a second dominantly inherited fragment

of 152-bp revealed co-segregation with the resistance

locus. The Xgwm469 fragment of 152-bp represents a

different locus than the 170-bp fragment that maps to

chromosome 6DS in the ITMI population (Roder

et al. 1998) (Figs. 1, 2). Since Xgwm272 mapped to

the long arm of chromosome 5D (Roder et al. 1998),

Mol Breeding (2009) 23:641–653 647

123

screening was conducted with an additional set of 32

EST-derived and genomic microsatellite markers that

mapped to this chromosome arm.

In all DH populations, genetic linkage between the

resistance locus and marker loci from the long arm of

wheat chromosome 5D was detected (Fig. 1). In total,

12 microsatellite loci were integrated into the genetic

map of the Savannah 9 Claire population and 15

microsatellite loci in the consensus map of the

Tremie-derived populations. Five common marker

loci were in collinear order between these two maps

confirming the common position of the SBCMV

resistance locus in cultivars Tremie and Claire.

Deviation of co-linearity in marker order between

two maps could be explained by the fact that the

Tremie-derived map was the consensus of three

populations. To identify additional closely linked

genomic fragments, resistant and susceptible

SBCMV bulks were screened with 256 EcoRI ?

3/MseI ? 3 AFLP primer combinations. Out of 23

AFLP fragments that were polymorphic between

DNA bulks, only one was mapped to 5DL. However,

in the Savannah 9 Claire population the AFLP

E37M49 was the closest proximal marker to the

SBCMV resistance locus. The closest to the resis-

tance locus marker in both populations was Xgwm469

(Fig. 1a, b). This marker co-segregated with the

resistance locus in the Tremie-derived population,

while in the Claire-derived population it mapped

1 cm distal to the resistance locus. To clarify the

position of Xgwm469-5D in relation to a putative

alternative source of SBCMV resistance this marker

was mapped in a population of 204 DH lines derived

from the Avalon 9 Cadenza cross. In this population

Xgwm469-5D co-segregated with Sbm1 (Fig. 1c).

Since the SBCMV resistance locus in Tremie was

located in the same chromosomal position as the

Sbm1 locus, which provides resistance to SBCMV in

the unrelated cv. Cadenza (Bass et al. 2006), it is

most likely that cultivars Tremie and Claire possess

Sbm1 or a gene allelic to Sbm1. However, since this

has not been proven by tests for allelism, the SBCMV

resistance locus in Tremie and Claire is temporarily

designated as Sbm Tremie/Claire.

For the physical assignment to a particular

segment of chromosome 5D, markers flanking the

resistance locus were tested on a set of 21 nulli-

tetrasomic aneuploid lines and the 5DL5 deletion

line. Markers proximal to the resistance locus, i.e.,

Xgpw343 and Xcfd10, as well as the distal marker

Xwmc765 were mapped to the same deletion bin

5DL5 (Fig. 3). As shown in Fig. 3, missing fragments

of flanking markers in lines 3 and 5, i.e., 5D and

5DL5, respectively, indicate the physical position to

Xcfd1830Xgwm8051Xgwm9314

Xgpw34315

Xcfd1035

E37M4939

SbmClaire48Xgwm46949Xwmc76551

Xbbrc14459

Xwmc44363

Xgwm27267

Xcfe30168

Xgpw2323

71

Xgdm630

Xgpw3433

Xgwm93115

Xgwm21222Xgdm133 Xbarc11024Xcfd1026Xcfd18327Xgwm80528

SbmTremie Xgwm46937Xwmc76539

Xwmc44347

Xgwm565 Xgwm269Xgwm65457

Xgdm1530

Xgwm21228

Xgdm11643

Xbarc11066

Xgwm469Sbm181

Xwmc76582Xbarc144 Xwmc44386

A B CFig. 1 Comparison of

genetic linkage maps for

wheat chromosome 5D

constructed using three

mapping populations:

Savannah 9 Claire (a),

Tremie-derived consensus

(b), Avalon 9 Cadenza (c).

SBCMV resistance locus is

indicated in bold.

Corresponding

microsatellite loci are

connected by thin lines

648 Mol Breeding (2009) 23:641–653

123

the sub-telomeric region of chromosome 5D. There-

fore, the SBCMV resistance locus is located on this

chromosome segment of 5DL.

Diagnostic value of SSR Xgwm469-5D

In the mapping study by Bass et al. (2006), genomic

microsatellite markers Xwmc765 and Xbarc110 were

the closest linked markers to Sbm1, whereas in our

study, screening of additional markers from chromo-

some 5D resulted in the identification of Xgwm469-

5D, Xgwm805, and AFLP E37M49, which are more

closely linked to the SBCMV-resistance locus in the

Savannah 9 Claire and Tremie-derived populations.

Since Xgwm469-5D and Xwmc765 were the two

closest makers to the SBCMV resistance locus in

three populations, the diagnostic value of these

markers was assessed in a collection of SBCMV

resistant and susceptible winter and spring wheat

cultivars originating from different parts of the world

(Table 2). In a set of 99 wheat cultivars Xgwm469-5D

and Xwmc765 detected three and twenty-two different

alleles, respectively. Although, Xwmc765 is located

only 1–3 cm distally from Xgwm469-5D it is of no

180

200

160

140

Cadenza

Claire and Tremie

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

Fig. 2 Molecular analysis of selected wheat genotypes with

the microsatellite marker GWM469 Xgwm469-5DL alleles

linked to SBCMV resistance are indicated by arrows. The

order of genotypes are: 1, Claire; 2, Cadenza; 3, F1 Claire 9

Cadenza; 4, Avalon; 5, Chinese Spring; 6, Ciano F67; 7, Aztek;

8, Tremie; 9, Soisson; 10, Savannah; 11, Axona; 12, Tonic; 13,

Sonora 64; 14, Wasp; 15, Moulin; 16, Flame. M, microSTEP-

20a (700) DNA ladder (Microzone Ltd). Due to extra 7-bp tail,

which was added to the 50 end of the reverse primer the

fragment size of Xgwm469-5D of 152 and 154-bp increased to

159 or 161-bp, respectively

Fig. 3 Assignment of SSR markers Xgpw343, Xcfd10, and

Xwmc765 flanking SbmClaire/Tremie to the chromosome deletion

bin 5DL5. The order of genotypes is: 1, N5A-T5D; 2, N5B-

T5D; 3, N5D-T5A; 4, Chinese Spring; 5, Chinese Spring

deletion line 5DL5, 6, water control. Upper band of Xgpw343maps to chromosome 5D, deletion bin 5DL5, while lower bandmaps to chromosome 5A. Single fragment of Xcfd10 maps to

chromosome 5D, deletion bin 5DL5. Upper fragment of

Xwmc765 maps to chromosome 5B while lowest fragment

maps to chromosome 5D, deletion bin 5DL5. At the left side of

the pictures is DNA ladder pUC19/MspI (Fermentas), while on

the right side of the pictures is the 1 kb DNA ladder

(Fermentas)

c

Mol Breeding (2009) 23:641–653 649

123

diagnostic value for the Sbm1 locus in comparison to

Xgwm469-5D. The Xgwm469-5D allele of 152-bp was

detected in Tremie and Claire as well as in other

resistant wheat cultivars having the resistant cv. Klein

Rendidor in their pedigree. In contrast, facultative

winter wheat cv. Cadenza, spring wheat cv. Tonic and

several related cultivars showed the Xgwm469-5D

allele of 154-bp. In addition, either the 152 or 154-bp

alleles of Xgwm469-5D were detected in a number of

old SBCMV-resistant wheat cultivars of primarily

non-European origin whose relationship to Tonic and

Cadenza or Tremie and Claire is unclear judging from

the available pedigree information. Importantly, in all

susceptible wheat cultivars, a third null allele of this

marker was observed. Therefore, Xgwm469-5D

appears to be an ideal diagnostic marker facilitating

the differentiation between the resistance derived from

cultivars Cadenza/Tonic and Tremie/Claire and the

identification of susceptible cultivars. These results are

in agreement with the pedigree analysis data which

suggest that cv. Cadenza is unrelated to Tremie or

Claire with respect to the donor of resistance. SBCMV

resistance in cultivars Claire and Tremie is most likely

derived from Klein Rendidor, whereas Sbm1 in cv.

Cadenza most likely originates from Sonora 64

(Fig. 4). Therefore, analysis of SBCMV resistance in

cultivars Tremie and Claire resulted in the identifica-

tion of closely linked markers with a high diagnostic

value that will facilitate efficient marker based selec-

tion procedures for SBCMV-resistance in wheat

breeding programs.

Discussion

It was suggested in earlier genetic studies from the

USA (Merkle and Smith 1983; Modawi et al. 1982)

that resistance to SBWMV in many US wheat

Axona----------Spring; null allele

Tonic------------Spring; 154bp

Maris Dove---

Hpg 522/66

H8810-47

Cadenza -------------Spring/Winter; 154bp

Heines Kolben

Sonora 64 Winter; 154bp

CardinalNo data

2772Mgh60-273

Parlo

Heines-Koga 2--

Raeckes White Chaff

Garnet

Heines Kolben

Cb-306-y-70 -------

Yecora 70 ------No data

Maris Hobbit (sib)

Moulin ------------Winter; 152bp

Maris Widgeon

Sib-32 No data

Tremie ----------Winter; 152bp

Siete Cerros 66

Klein RendidorSpring; 152bp

Sonora 64Winter; 154bp

Ciano F67Spring; null allele

Ciano F67Spring; null allele

Taurus

Flame ---Winter; 152bp

Waspnull allele

Moulin ---------Winter; 152bp

Claire -------------Winter; 152bp

Cb-306-y-70 -------

Yecora 70 ------No data

Maris Hobbit (sib)

Maris Widgeon

Ciano F67Spring; null allele

Siete Cerros 66

Klein RendidorSpring; 152bp

Sonora 64Winter; 154bp

Ciano F67Spring; null allele

Fig. 4 Pedigree

relationships of SBCMV

resistant varieties with

assignment of Xgwm469-

5DL donor alleles

650 Mol Breeding (2009) 23:641–653

123

cultivars is derived from the following old cultivars:

(1) the Argentinean cv. Klein Rendidor (this cultivar

is also an ancestor of European SBCMV-resistant

cultivars Tremie and Claire), (2) the US cv. Turkey,

and (3) the Ukrainian cv. Odesskaja 51. Therefore,

the number of detected alleles for Xgwm469-5D is

rather low for such a diverse set of germplasm tested,

but may be explained by the in general low diversity

of the D genome (Balfourier et al. 2007; Wicker et al.

2009). Interestingly, however, Xwmc765 displayed a

much higher allelic diversity (Table 2).

The 152 and 154-bp alleles were linked to

resistance and the null allele was linked to suscep-

tibility in all genotypes tested, suggesting that the

SBCMV-resistance existing in the wheat germplasm

tested may have been derived from two separate

sources. However, further studies (e.g., an allelism

test or the isolation of the SBCMV resistance locus),

will be required to clarify whether Sbm1 in cv.

Cadenza is allelic to the SBCMV resistance locus in

cultivars Tremie and Claire. In addition, several

genotypes with intermediate phenotypes were iden-

tified in the studied collection and very rarely also

within the segregating populations. Occurrence of

intermediate phenotypes after visual scoring suggests

the presence of a major gene as well as other genes

with minor effects contributing to the resistance,

possibly acting as suppressors of resistance. How-

ever, it is also important to mention that the

‘translocation resistance’ is highly influenced by the

environment and the root-shoot translocation block

can be occasionally overcome by the virus (Lyons

et al. 2008; Myers et al. 1993). The exact factors

contributing to the breakdown of translocation resis-

tance are as yet unknown. However, it is conceivable

that the translocation resistance can be overcome

either under high inoculum pressure (i.e., when an

excessively high number of viruliferous P. graminis

zoospores is present close to the plant’s root system)

or when the plant is suffering from other biotic (i.e.,

infection by other phytopathogens) or abiotic stress

(i.e., freezing).

It is interesting to note that although genetically

quite diverse wheat germplasm was tested in this

study, that only three different alleles were detected

at the Xgwm469-5D locus. This number is much

lower than that found in other wheat genotyping

studies with microsatellite markers (Balfourier et al.

2007; Huang et al. 2002; Roder et al. 1998). The low

level of polymorphism at the Xgwm469-5D locus

may be because the wheat D-genome is known to

exhibit a generally low level of polymorphism

(Balfourier et al. 2007; Wicker et al. 2009). It is also

possible that this region on 5DL contains some

(unknown) genes that are essential for crop growth,

development or performance and, therefore, is sub-

jected to a strong purifying selection pressure. A low

level of polymorphism was also detected in barley at

the Bmac29 locus which is closely linked to the

bymovirus resistance locus rym4/rym5 (Graner et al.

1999) and used in marker assisted selection (MAS)

since 1999 (Ordon et al. 2004). Such microsatellite

loci, which are closely linked to the genes of interest

and display low allelic diversity, are best suited for

MAS procedures as they can be reliably used in

breeding programs involving many diverse crosses.

In this study, we identified a previously unmapped

microsatellite locus Xgwm469 as the most closely

linked to the SBCMV resistance gene on 5DL. Here

we also provide evidence that GWM469 is well

suited for use in MAS for reliable selection of

resistant genotypes at early developmental stages and

on a single plant level. This is of special importance

as in many European countries uniformly infested

fields required for reliable resistance screening of

breeding materials are not available. Therefore, the

availability of the GWM469 marker will facilitate

quarantine breeding, i.e., the development of resistant

cultivars in the absence of the virus. This is especially

relevant due to the risk of rapid spreading of these

soil-borne viruses, as seen previously for the soil-

borne viruses of barley (Barley yellow mosaic virus

and Barley mild mosaic virus) in Germany (Huth

1989). However, GWM469 also has some drawbacks.

First, in contrast to many microsatellites, it is a

dominant marker that does not allow the selection of

homozygous resistant genotypes in standard (i.e., not

doubled haploid) segregating heterozygous popula-

tions. In future, this could be of minor importance

when more European wheat breeders will use

doubled haploid populations in which due to homo-

zygosity dominant markers are as informative as co-

dominant ones. Also, null alleles derived from

susceptible lines cannot be distinguished from an

absence of amplification due to a PCR failure. One

way to overcome this would be to use an internal

standard such as an additional monomorphic marker.

In our case, the major fragment of Xgwm469 of

Mol Breeding (2009) 23:641–653 651

123

170-bp is well suited as an internal control of the

PCR reaction. However, the best marker remains the

gene itself since even when the marker is tightly

linked to the gene, the recombination may still occur.

Therefore, the flanking markers developed in this

study may serve as the starting point for the

construction of a high resolution map finally leading

to the map based isolation of Sbm1. When the

resistance gene sequence is revealed it should then be

possible to develop robust allele-specific markers for

reliable and errorless identification of resistant geno-

types in the laboratory.

Acknowledgments The work is supported by a grant in the

European Community’s Sixth Framework Program

WHEATPROTECT (EU contract number COOP-CT-2004-

512703). These results and this publication reflect only the

author’s view and EU is not liable for any use that may be made

of the information contained in this article. The authors like to

thank Prof. B. Gill (Kansas State University, USA) for kindly

providing seeds of NT and 5DL5 Chinese Spring lines, Dr. L.

Sayers (John Innes Centre, UK) for providing Avalon 9

Cadenza DH mapping population, and various European wheat

breeding companies as well as Dr. H. E. Bockelman (National

Small Grains Collection, USDA Aberdeen, USA) and Dr. R.

Singh (CIMMYT, Mexico) for providing wheat genotypes used

in disease resistance tests and/or molecular genetic analyses. We

are also grateful to Dr. R. Bayles (NIAB, Cambridge, UK) and

Mr. M. Oatley (Paxcroft Farms, Trowbridge, UK) for letting us

sample SBCMV-infested soil in Wiltshire site. We would also

like to thank Ms. B. Knupfer and M. Weilepp for excellent

technical assistance. Rothamsted Research receives grant-aided

support from the Biotechnology and Biological Sciences

Research Council of the United Kingdom.

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