q NIAB 2007 ISSN 1479-2621
Plant Genetic Resources: Characterization and Utilization 5(2); 100 –103 DOI: 10.1017/S1479262107672311
Short communication
The use of simple sequence repeat (SSR) markers to identify and map alien segments carrying genes for effective resistance to leaf rust in bread wheat Nayyer Iqbal†, Firdissa Eticha‡, Elena K. Khlestkina§, Annette Weidner, Marion S. Ro¨der and Andreas Bo¨rner* Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany
Received 16 October 2006; Accepted 20 November 2006
Abstract Aegilops markgrafii is a useful source of genes encoding both resistance to biotic stress and high seed lysine content. Bread wheat/Ae. markgrafii introgression lines expressing leaf rust resistance were developed from a cross between a leaf rust-resistant Ae. markgrafii accession and the susceptible bread wheat cultivar ‘Alcedo’. The content of introgressed segments present in five sister introgression lines was assessed with the help of chromosome-specific simple sequence repeats (SSRs). One of the lines was used as a parent of a 140 individual F2 mapping population, by crossing with the leaf rust-susceptible bread wheat cv. ‘Borenos’. The population was tested for susceptibility or resistance to leaf rust, and linkage analysis indicated the presence of a quantitative trait locus (QLr.ipk-2A) originating from the Ae. markgrafii parent, mapping to the distal segment of chromosome arm 2AS.
Keywords: Aegilops markgrafii; disease resistance; interspecific introgression lines; microsatellite markers; Puccinia recondita; Triticum aestivum
Wheat suffers from a number of pathogens, and one of the most important of its foliar fungal diseases is leaf rust. More than 50 leaf rust resistance (Lr) genes have been documented (McIntosh et al., 2003), but most have been overcome by the pathogen. Wild relatives
* Corresponding author. E-mail:
[email protected] † Present address: Nuclear Institute for Agriculture and Biology (NIAB), Jhang Road, Faisalabad, Pakistan. ‡ Present address: Institute of Plant Breeding, University of Natural Resources and Live Sciences, Gregor Mendel Str. 33, 1180 Vienna, Austria. § Present address: Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentyeva ave. 10, Novosibirsk, 630090, Russia.
and related species represent a valuable source of genes for crop improvement, and more than 20 Lr genes have been introduced into bread or durum wheat from a range of species (Schachermayr et al., 1995; Friebe et al., 1996; Cenci et al., 1999; Xie et al., 2003; Leonova et al., 2004). The diploid Aegilops markgrafii (syn. Ae. caudata; genome CC) is a valuable source of genes encoding resistance to powdery mildew, leaf rust and stripe rust (Schubert et al., 1995), and is therefore a good candidate to be a donor of exotic alleles and genes of utility for wheat improvement. Microsatellite (simple sequence repeat, SSR) loci are associated with significant levels of sequence polymorphism. A growing number of SSR loci have been incorporated
SSR markers and resistance to leaf rust in wheat
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into the wheat genetic map (Ro¨der et al., 1998; Somers et al., 2004). The specificity of these markers can help to determine which chromosome(s) are involved in alien introgression materials (Peil et al., 1998; Iqbal et al., 2000). We therefore used SSRs to characterize a set of wheat/Ae. markgrafii introgression lines.
Experimental Five leaf rust-resistant sister introgression lines, which all shared a wheat-like growth habit, were selected from a cross between the leaf rust-susceptible wheat cultivar ‘Alcedo’ and the resistant Ae. markgrafii accession ‘S740-69’ (Schubert, 2001; Weidner, 2004). One of these (N43) was crossed to the susceptible wheat cultivar ‘Borenos’, and the resulting segregating 140 member F2 population was used for genetic mapping. A pathogenicity test was performed on seedlings at the two-leaf stage in a growth chamber. The leaf rust inoculum carried virulences against Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3bg, Lr3ka, Lr10, Lr11, Lr13, Lr14a, Lr14b, Lr15, Lr16, Lr17, Lr18, Lr20, Lr21, Lr23, Lr26, Lr28, Lr30, Lr32, Lr33, Lr37, Lr38 and Lr44. After a 24 h dark incubation at 14–158C and 100% humidity, the temperature was reset to 208C, and permanent light and normal humidity was maintained. After 10 days, the plants were scored for the intensity of leaf rust infection, using a measurement scale of 0
2A
QLr.ipk -2A
1.7 4.3 5.9
2B Xgwm1176 Xgwm497 Xgwm614 Xgwm636 Xgwm1053 Xgwm830 Xgwm296
(leaves without any visible symptom) to 4 (clearly visible red-brown pustules) (McIntosh et al., 1995). For SSR analysis, DNA was isolated from individual seedling leaves and PCR reaction conditions were as described by Ro¨der et al. (1998). The profiles were generated by an Automated Laser Fluorescence (ALF) express semi-automatic DNA sequencing device. A set of 226 SSR loci of known intrachromosomal location (Ro¨der et al., 1998; unpublished data) were tested. Of these, four (Xgwm614, Xgwm636, Xgwm497 and Xgwm1176), all mapping to chromosome arm 2AS, were absent from the introgression lines, as were Xgwm148, Xgwm374 and Xgwm972 (2BS), Xgwm160 (4AL) and Xgwm732 and Xgwm1103 (6DL). The GWM830 (2AL), GWM1053 (2AL) and GWM1005 (3BL) primer pairs also amplified Ae. markgrafii alleles, replacing the wheat alleles at these loci. These results are graphically illustrated in Fig. 1. Only 19 of the 140 F2 plants were leaf rust-susceptible, a highly significant deviation from the ratio expected for a monogenic trait. The segregation of SSR loci located on chromosome arm 2A was also highly distorted, although in contrast, the segregation of the other SSR loci detecting the presence of an alien introgression was consistent with the expected 3:1 ratio (Table 1). Simple linkage calculation between leaf rust resistance and SSR loci was hindered by the segregation distortion, and so a quantitative trait locus (QTL) approach was attempted. This resulted
3B
4A
6D
Xgwm257 Xgwm148 Xgwm374 Xgwm972 Xgwm630
c
Xgwm108 Xgwm1005 Xgwm112b
Xgwm742 Xgwm160
Xgdm98 Xgwm732 Xgwm1103 Xksud27a
Xgwm1179b
Fig. 1. The characterization of wheat/Aegilops markgrafii introgression lines. Alien segments indicated as dark zones, with corresponding and flanking markers shown. Underlined markers (on chromosome 2A) were those employed for linkage analysis. Genetic distances in cM. c, centromere.
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Table 1. Segregation patterns for leaf rust resistance and SSR loci in an F2 population derived from the cross introgression line £ normal bread wheat Locus Lr Xgwm1176 Xgwm497 Xgwm636 Xgwm1053 Xgwm830 Xgwm148 Xgwm374 Xgwm1005 Xgwm855 Xgwm742 Xgwm160 Xgwm1103
Chromosome
Expected
Observed
x2 value
2AS 2AS 2AS 2AS 2AS 2AS 2BS 2BS 3BL 4AL 4AL 4AL 6DL
35:105 35:105 35:105 35:105 105:35 105:35 35:105 35:105 35:105 35:105 35:105 35:105 35:105
19:121 21:119 21:119 20:120 90:50 86:54 36:104 40:100 35:105 30:110 32:108 30:110 28:112
9.75** 7.47** 7.47** 8.57** 8.57** 13.78** 0.04 ns 0.95 ns 0.00 ns 0.95 ns 0.34 ns 0.95 ns 1.87 ns
** P , 0.01. ns, not significant.
in the identification of a significant QTL (LOD score 5.14) on chromosome arm 2AS (Table 2 Fig. 1), designated QLr.ipk-2A.
Discussion The homoeologous relationships between the A, B, D genomes of wheat and the C genome of Ae. markgrafii have been described by Peil et al. (1998) and Schubert (2001). Although the authors have shown that there are rearrangements between the wheat and Aegilops genomes, recombination may occur. Chromosome B of Ae. markgrafii carries gene(s) for resistance to Puccinia recondita and this chromosome shares homoeology with those in groups 2 and 4 of wheat (Peil et al., 1998; Schubert, 2001). The SSR analysis of the introgression lines was consistent with the presence of Ae. markgrafii segments on homoeologous group 2 and 4 chromosomes, as well as detecting additional transfers involving the long arms of chromosomes 3B and 6D. Distorted segregation ratios are commonplace in mapping populations. Gametocidal effects of chromosomes derived from Aegilops species have been described earlier (Endo, 1983, 1988, 1996). The gametocidal action of Table 2. QTL mapping of leaf rust resistance with respect to SSR loci Marker Xgwm1053 Xgwm830 Xgwm1176 Xgwm614 Xgwm636 Xgwm497 * P , 0.05.
F value
R2
LOD score
25.40 24.26 1.04 1.04 1.04 1.04
0.1526 0.1468 0.0147 0.0147 0.0147 0.0147
5.14* 4.93* 0.46 0.46 0.46 0.46
Ae. markgrafii chromosomes was reported by Endo and Katayama (1978). Therefore, it is possible that the segregation distortion observed in the present mapping population is caused by the activity of such a gametocidal gene. An alternative explanation for the distortion may be a poor level of compensation between the introduced segment and the substituted wheat segment. Of the more than 65 Lr loci (both major genes and QTLs) described to date (McIntosh et al., 2003), about 15 originate from Aegilops species. However, this is the first report of a resistance locus from Ae. markgrafii. The potential of this species as a donor for disease resistance (including leaf rust) was already well understood by the 1980s (Frauenstein and Hammer, 1985; Valkoun et al., 1985) and so the species should be considered to be a strong candidate as a source of exotic genes to improve the disease resistance of advanced wheat germplasm.
Acknowledgements Nayyer Iqbal thanks the Alexander von Humboldt Foundation (grant no. PAK/1117127 STP) for financial support.
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