Abstract
Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease of wheat (Triticum aestivum) in China and worldwide, causing severe yield losses annually. Wild emmer (T. dicoccoides) accession IW72 collected from Israel is resistant to powdery mildew at the seedling and adult stages. Genetic analysis indicated that the resistance was controlled by a single dominant gene, temporarily designated MlIW72. The F2 population and F3 families derived from a hybrid between IW72 and susceptible durum wheat line Mo75 were used for molecular mapping of the resistance gene. MlIW72 was linked with SSR loci Xgwm344, Xcfa2040, Xcfa2240, Xcfa2257 and Xwmc525 on the long arm of chromosome 7A. In addition, two STS markers, MAG2185 (derived from RFLP marker PSR680) and MAG1759 (developed from EST CD452874), were mapped close to MlIW72. All these markers were physically located in the terminal bin 0.86–1.00 of 7AL. The chromosome location and genetic mapping results suggested that the powdery mildew resistance gene identified in wild emmer accession IW72 might be a new allele at the Pm1 locus or a new locus closely linked to Pm1.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most devastating diseases of wheat (Triticum aestivum, AABBDD; 2n = 42) in China and worldwide and severe epidemics often occur in areas with cool and humid climates, causing significant yield losses. Compared with fungicides, resistant cultivars are cost-effective and environmentally safe approach to control the disease. However, the race-specific resistance genes commonly used are often overcome by new races possessing corresponding virulence genes in relatively short time periods. Thus, it is necessary to discover more diverse resistance genes to enrich those available for resistance breeding. About 50 formally designated Pm resistance alleles occur at 32 loci (Huang and Röder 2004; McIntosh et al. 2003, 2004, 2005; Miranda et al. 2006, 2007). In addition, there are several temporarily designated resistance genes (Qiu et al. 2005; Yao et al. 2007; Zhu et al. 2005; Zhou et al. 2005; Mohler et al. 2005). Many of the resistance genes were introduced from, or found in, wheat relatives.
Wild emmer, Triticum turgidum var. dicoccoides [T. dicoccoides, (AABB; 2n = 28)], the ancestor of cultivated wheat, has great potential for wheat improvement (Nevo et al. 2002), being a genetic resource of genes for quality and grain protein content (Nevo et al. 2002; Xu et al. 2004; Uauy et al. 2006), as well as pathogen resistance (Gerechter-Amitai and Stubbs 1970; Moseman et al. 1984, 1985; Nevo et al. 1991; Buerstmayr et al. 2003). Wild emmer genotypes originating from Israel were highly resistant to wheat powdery mildew in China (Xie et al. 2003). Several resistance genes were identified in, and transferred from, wild emmer to common wheat (Reader and Miller 1991; Rong et al. 2000; Liu et al. 2002; Mohler et al. 2005).
Molecular markers, such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNAs (RAPDs) and microsatellites or simple sequence repeats (SSRs), have been widely used and have greatly accelerated progress in wheat genetic resources and breeding. Molecular markers for more than 30 powdery mildew resistance genes/alleles in wheat have been reported (Huang and Röder 2004; McIntosh et al. 2003, 2004, 2005; Zhu et al. 2005; Miranda et al. 2006, 2007; Yao et al. 2007). Pm3b was successfully isolated by map-based cloning with the aid of closely linked molecular markers (Yahiaoui et al. 2004).
The present paper reports the genetic analysis and molecular mapping of a powdery mildew resistance gene in one of these Israeli wild emmer genotypes highly resistant to powdery mildew in China.
Materials and methods
Plant materials
Wild emmer accession IW72 (collected in Kokhav-Hashahar, Israel, the original accession number was TZ74) was highly resistant to powdery mildew (no visible disease symptoms). A susceptible durum wheat line Mo75 was kindly provided by Prof. Xiao Chen (Institute of Crops, China Academy of Agricultural Science). An F2 population and derived F3 families from Mo75/ IW72 were used for genetic analysis and mapping.
Evaluation of powdery mildew resistance
Bgt isolate E09, virulent to Pm1a, Pm3a, Pm5a, Pm7 and Pm8, but avirulent to IW72, was used for disease response tests. Two hundred and five F2 plants were evaluated at the adult stage (from heading to grain filling) in the inoculated nursery for response to powdery mildew. Plants with no visible disease symptoms were classified resistant, whereas those with freely sporulating lesions were regarded as susceptible. The derived F3 progenies were tested at the seedling stage in a greenhouse to confirm the phenotypes of the F2 plants and to establish the genotypes. Fifteen to 20 seedlings of each F3 family were inoculated at the one-leaf stage by brushing conidia from neighboring sporulating susceptible seedlings of cv. Xuezao on to fully expanded first leaves. The results were recorded two weeks after inoculation when the Xuezao control and susceptible test seedlings showed distinct mildew symptoms. Reactions were scored on a 0, 0, and 1 to 4 infection type (IT) scale, with 0 representing no visible symptoms, 0; representing necrotic flecks, and 1, 2, 3, 4 for highly resistant (necrosis with low sporulation), resistant (necrosis with medium sporulation), susceptible (no necrosis with medium to high sporulation), and highly susceptible (no necrosis with full sporulation) reactions, respectively.
Molecular marker analysis
Total DNA was extracted from leaf tissue samples of each F2 adult plant following the method of Song and Henry (1995). Two DNA pools were created by combining equal amounts of DNA from 8 resistant or 8 susceptible F2 plants for bulked segregant analysis (BSA) (Michelmore et al. 1991).
Wheat microsatellite markers (gwm, wmc, cfa, and cfd series) mapped to the A and B genomes were chosen for marker analysis. Relevant information for these markers is published on the GrainGenes website (http://www.wheat.pw.usda.gov). In addition, STS markers (Yao et al. 2007), that were closely linked to Pm1a and other powdery mildew resistance genes located on chromosome 7AL in einkorn wheat, were also tested.
PCR was performed in a volume of 10 μl containing 50 ng of genomic DNA, 10 mM Tris-Hcl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of dNTPs, 20 ng of each primer pair, and 0.75 U Taq DNA polymerase. The amplification programs were as follows: 94°C for 3 min, followed by 40 cycles of 94°C for 1 min, 50–60°C (depending on the specific primers) for 1 min, 72°C for 2 min and a final extension at 72°C for 10 min. The PCR products were separated in 8% non-denaturing polyacrylamide gels (39 acrylamide : 1 bisacrylamide ). Gels were then silver-stained (Xu et al. 2002) and photographed.
Chromosomal arm and physical bin assignments of markers
The markers linked to the powdery mildew resistance were tested on DNA samples of Chinese Spring homoeologous group 7 nullisomic-tetrasomics and ditelosomics to assign chromosome and chromosome arm locations. Four Chinese Spring 7AL deletion lines (d7AL-1 FL0.39, d7AL-16 FL0.86, d7AL-17 FL0.71, d7AL-21 FL0.74) were used to physically locate the markers. Each marker was mapped to a chromosome bin flanked by breakpoints of the largest deletion possessing the fragment and the smallest deletion lacking it after comparing the amplification patterns (Qi et al. 2003; Sourdille et al. 2004).
Data analysis
Chi-squared tests were used to determine the goodness-of-fit of observed data with expected segregation ratios. Linkage between molecular markers and the resistance gene was analyzed using Mapmaker 3.0, with a LOD score of 3.0 as threshold.
Results
Genetic analysis of the powdery mildew resistance in wild emmer accession IW72
IW72 was highly resistant to Bgt isolate E09 (IT 0 or 0;). Of 205 F2 plants from Mo75/IW72, 154 were resistant and 51 were susceptible at the adult stage, indicating single locus segregation (χ2 3:1 = 0.0016, df = 1, P > 0.95). This segregation was further confirmed by progeny seedling tests. Among 154 F3 lines derived from resistant F2 plants, 47 were homozygous resistant, 107 segregated, and 51 susceptible F2 plants were homozygous susceptible (χ2 1:2:1 = 0.583, df = 2, P > 0.75). The resistance gene was temporarily designated MlIW72.
Mapping of MlIW72
Initially, 28 wheat SSR markers mapping to the A and B genomes were screened for polymorphism between the resistant and susceptible DNA pools. One marker, GWM344, was polymorphic between the pools. Xgwm344 was found co-segregating with MlIW72 (Fig. 1). On the wheat microsatellite maps constructed by Röder et al. (1998) and Peng et al. (2002), Xgwm344 appears on the long arms of both chromosomes 7B and 7A, then other SSR markers located on 7AL and 7BL were tested for polymorphism, and four of them (CFA2040, CFA2240, CFA2257 and WMC525) detected polymorphisms between the resistant and susceptible pools. All four co-dominant loci, Xcfa2040, Xcfa2240, Xcfa2257 and Xwmc525, were also co-segregated with MlIW72.
Five STS primer pairs of markers linked to powdery mildew resistance genes located on chromosome 7AL in einkorn wheat reported by Yao et al. (2007) were screened for polymorphism between IW72 and Mo75, and the resistant and the susceptible DNA pools. Two, MAG1759 and MAG2185, were co-segregated with MlIW72 (Fig. 2). MAG1759 showed a co-dominant inheritance pattern whereas MAG2185, amplifying a 240 bp polymorphic fragment, was a coupling-phase dominant marker closely associated with the resistance allele.
Linkage analysis showed that MlIW72 was flanked by Xmag2185 and Xmag1759 with genetic distances of 3.3 cm and 8.2 cm, respectively (Fig. 3a). The linked microsatellite loci Xgwm344, Xcfa2240, Xcfa2257, Xwmc525 and Xcfa2040 were at greater distances from MlIW72.
A set of Chinese Spring homoeologous group 7 nullisomic-tetrasomics and ditelosomics were used to assign the chromosomal locations of the molecular marks. All except Xmag2185, which had no corresponding PCR band in Chinese Spring, were absent in N7AT7B, N7AT7D, and Dt7AL, indicating they were located on the long arm of chromosome 7A. These results were further confirmed when four Chinese Spring 7AL deletion lines (d7AL-1 FL0.39, d7AL-16 FL0.86, d7AL-17 FL0.71, d7AL-21 FL0.74) were used to determine the sub-chromosomal region harbouring the markers. No amplification products of the markers were observed in the deletion lines indicating their physical locations in chromosome 7AL distal bin 0.86–1.00 (Fig. 4). We concluded that MlIW72 was distally located in chromosome 7AL (Fig. 3b).
Discussion
A dominant powdery mildew resistance gene effective at both the seedling and adult stages was identified in wild emmer accession IW72. Molecular mapping indicated that MlIW72 was physically located in terminal bin 0.86–1.00 of chromosome 7AL, close to several markers including Xgwm344, Xmag2185 and Xmag1759 (Fig. 3).
Several different powdery mildew resistance genes have been located on chromosome 7AL; these include Pm1a, Pm1e, Pm9 and mlRD30 originating from common wheat (Hsam et al. 1998; Singrün et al. 2003, 2004; Schneider et al. 1991), Pm1b and likely Pm1c transferred from T. monococcum (Hsam et al. 1998), Pm1d derived from T. spelta (Hsam et al. 1998), PmU introduced from T. urartu (Qiu et al. 2005), and Mlm2033 and Mlm80 identified in T. monococcum (Yao et al. 2007). Srnic et al. (2005) also located two partially dominant major genes transferred to winter wheat from T. monococcum and T. timopheevii in chromosome 7AL. MlIW72 is the first reported Pm gene in T. dicoccoides to be located on chromosome 7AL. Thus chromosome 7AL in common wheat and its relatives appears to be an important region for powdery mildew resistance genes.
Singrün et al. (2003) found that Pm22 and Pm1c were both closely linked to Xgwm344-7A and demonstrated that the earlier named Pm22 was an allele (Pm1e) of the complex Pm1 locus. Yao et al. (2007) mapped two powdery mildew resistance genes, Mlm2033 and Mlm80, on the long arm of chromosome 7A in einkorn. These genes were closely linked to Xgwm344, Xmag2185 and Xmag1759, and suggesting a likely association with the Pm1 locus. In the present study, MlIW72 was located to a similar chromosome region providing additional evidence for a complex Pm1 site.
The present work adds to the potential of wild emmer for wheat improvement (Nevo et al. 2002). Potentially useful genes found in wild emmer can be readily transferred to common wheat by direct hybridization, backcrossing and selection.
References
Buerstmayr H, Stierschneider M, Steiner B et al (2003) Variation for resistance to head blight caused by Fusarium graminearum in wild emmer (Triticum dicoccoides) originating from Israel. Euphytica 130:17–23
Gerechter-Amitai ZK, Stubbs RW (1970) A valuable source of yellow rust resistance in Israeli populations of wild emmer, Triticum dicoccoides Koern. Euphytica 19:12–21
Hsam SLK, Huang XQ, Ernst F et al (1998) Chromosomal location of genes for resistance to powdery mild ew in common wheat (Triticum aestivum L. em Thell.) 5 alleles at the Pm1 locus. Theor Appl Genet 96:1129–1134
Huang XQ, Röder MS (2004) Molecular mapping of powdery mildew resistance genes in wheat: A review. Euphytica 137:203–223
Liu ZY, Sun QX, Ni ZF et al (2002) Molecular characterization of a novel powdery mildew resistance gene Pm30 in wheat originating from wild emmer. Euphytica 123:21–29
Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating population. Proc Natl Acad Sci USA 88:9828–9832
McIntosh RA, Yamazaki Y, Devos KM et al (2003) Catalogue of gene symbols for wheat. In: Pogna NE, Romano M, Pogna EA, Galterio G (eds) Proceedings of 10th International Wheat Genetics Symposium, vol 4. Instituto Sperimentale per la Cerealicoltura, Rome, Italy, pp 1–34
McIntosh RA, Devos KM, Dubcovsky J et al (2004) Catalogue of gene symbols for wheat: 2004 (suppl). http://www.wheat.pw.usda.gov/GG2/pubs.shtml
McIntosh RA, Devos KM, Dubcovsky J et al (2005) Catalogue of gene symbols for wheat: 2005 (suppl). http://www.wheat.pw.usda.gov/GG2/pubs.shtml
Miranda LM, Murphy JP, Marshall D et al (2006) Pm34: A new powdery mildew resistance gene transferred from Aegilops tauschii Coss. to common wheat (Triticum aestivum L.). Theor Appl Genet 113:1497–1504
Miranda LM, Murphy JP, Marshall D et al (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
Mohler V, Zeller FJ, Wenzel G et al (2005) Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.) 9 Gene MlZec1 from Triticum diccoides-derived wheat line Zecoi-1. Euphytica 142:161–167
Moseman JG, Nevo E, El-Morshidy MA et al (1984) Resistance of Triticum dicoccoides collected in Israel to infection with Erysiphe graminis tritici. Euphytica 33:41–47
Moseman JG, Nevo E, Gerechter-Amitai ZK et al (1985) Resistance of Triticum dicoccoides collected in Israel to infection with Puccinia recondita tritici. Crop Sci 25:262–265
Nevo E, Gerechter-Amitai ZK, Beiles A (1991) Resistance of wild emmer wheat to stem rust: Ecological, pathological and allozyme associations. Euphytica 53:121–130
Nevo E, Korol AB, Beiles A et al (2002) Evolution of wild emmer and wheat improvement population genetics, genetic resources, and genome organization of wheat’s progenitor, Triticum dicoccoides. Springer Verlag, Heidelberg
Peng JH, Korol AB, Fahima T et al (2002) Molecular genetic maps in wild emmer wheat, Triticum dicoccoides: Genome-wide coverage, massive negative interference, and putative quasi-linkage. Genome Res 10:1509–1531
Qi L, Echalier B, Friebe B et al (2003) Molecular characterization of a set of wheat deletion stocks for use in chromosome bin mapping of ESTs. Funct Integr Genomics 3:39–55
Qiu YC, Zhou RH, Kong XY et al (2005) Microsatellite mapping of a Triticum urartu Tum derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.). Theor Appl Genet 111:1524–1531
Reader SM, Miller TE (1991) The introduction into bread wheat of a major gene for resistance to powdery mildew from wild emmer wheat. Euphytica 53:57–60
Röder MS, Korzun V, Wendehake K et al (1998) A microsatellite map of wheat. Genetics 149:2007–2023
Rong JK, Millet E, Manisterski J et al (2000) A new powdery mildew resistance gene: Introgression from wild emmer into common wheat and RFLP-based mapping. Euphytica 115:121–126
Schneider DM, Heun M, Fischbeck G (1991) Inheritance of the powdery mildew resistance gene Pm9 in relation to Pm1 and Pm2 of wheat. Plant Breed 107:161–164
Singrün C, Hsam SLK, Hartl L et al (2003) Powdery mildew resistance gene Pm22 in cultivar Virest is a member of the complex Pm1 locus in common wheat (Triticum aestivum L. em Thell.). Theor Appl Genet 106:1420–1424
Singrün C, Hsam SLK, Zeller FJ et al (2004) Localization of a novel recessive powdery mildew resistance gene from common wheat line RD30 in the terminal region of chromosome 7AL. Theor Appl Genet 109:210–214
Song W, Henry RJ (1995) Molecular analysis of the DNA polymorphism of wild barley (Hordeum spontaneum) germplasm using the polymerase chain reaction. Genet Resour Crop Evol 42:273–280
Sourdille P, Singh S, Cadalen T et al (2004) Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (Triticum aestivum L.). Funct Integr Genomics 4:12–25
Srnic G, Murphy JP, Lyerly JH et al (2005) Inheritance and chromosomal assignment of powdery mildew resistance genes in two winter wheat germplasm lines. Crop Sci 45:1578–1586
Uauy C, Distelfeld A, Fahima T et al (2006) A NAC Gene Regulating Senescence Improves Grain Protein, Zinc, and Iron Content in Wheat. Science 314:1298–1301
Xie C, Sun Q, Yang Z (2003) Resistance of wild emmers from Israel to wheat rusts and powdery mildew at seedling stage (in Chinese). Acta Tritical Crops 23:39–42
Xu S, Tao Y, Yang Z et al (2002) A simple and rapid methods used for silver staining and gel preservation. Hereditas (Beijing) 24:335–336
Xu SS, Khan K, Klindworth DL et al (2004) Chromosomal location of genes for novel glutenin subunits and gliadins in wild emmer wheat (Triticum turgidum L. var. dicoccoides). Theor Appl Genet 108:1221–1228
Yahiaoui N, Srichumpa P, Dudler R et al (2004) Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b form hexaploid wheat. Plant J 37:528–538
Yao G, Zhang J, Yang L et al (2007) Genetic mapping of two powdery mildew resistance genes in einkorn (Triticum monococcum L.) accessions. Theor Appl Genet 114:351–358
Zhou R, Zhu Z, Kong X et al (2005) Development of wheat near-isogenic lines for powdery mildew resistance. Theor Appl Genet 110:640–648
Zhu ZD, Zhou RH, Kong XY et al (2005) Microsatellite markers linked to two genes conferring resistance to powdery mildew in common wheat introgressed from Triticum carthlicum acc PS5. Genome 48:585–590
Acknowledgements
The authors thank Dr. Robert McIntosh for his comprehensive review of this manuscript. This work was financially supported by the National Natural Science Foundation of China (30571151), National Fund for Distinguished Young Scholars (30425039), State High-Tech Programs (2006AA100102, 2006AA10Z1C4, 2006AA10Z1E9) Fok Ying Tung Education Foundation (94021), and Beijing Natural Science Foundation (6061003).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ji, X., Xie, C., Ni, Z. et al. Identification and genetic mapping of a powdery mildew resistance gene in wild emmer (Triticum dicoccoides) accession IW72 from Israel. Euphytica 159, 385–390 (2008). https://doi.org/10.1007/s10681-007-9540-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10681-007-9540-1