Introduction

Powdery mildew is a foliar disease of wheat caused by Blumeria graminis f. sp. tritici (Bgt). Due to potential high yield losses and decreased grain quality, powdery mildew is an economically important wheat disease worldwide in the cool and semi-humid wheat growing areas. Breeding for resistance is the most profitable and environmentally acceptable strategy to control powdery mildew. More than 60 powdery mildew resistance genes located at 41 loci (Pm1-Pm45, Pm18 = Pm1c, Pm22 = Pm1e, Pm23 = Pm4c, Pm31 = Pm21) (Ma et al. 2011; Hsam et al. 1998; Singrün et al. 2003; Hao et al. 2008; Xie et al. 2011) have been identified and designated in wheat and its wild relatives.

A significant problem in wheat breeding and production is the loss of resistance caused by virulent races. Therefore, it is urgent to search for new powdery mildew resistance genes. In order to improve resistance, wheat (Triticum aestivum L.) had been crossed with its related genera (Jiang et al. 1993), such as Aegilops, Elytrigia, Secale, Haynaldia, and related species of Triticum, for instance, T. boeoticum, T. dicoccoides, T. carthlicum and T. timopheevii, which represent a reservoir of genes for resistance to multiple diseases. Intergeneric and interspecific crosses have resulted in the transfer of desirable fungal resistance into wheat; for example, powdery mildew resistance genes, Pm7, Pm8, Pm17, and Pm20 originated from Secale (McIntosh et al. 2011); Pm12, Pm13, Pm19, Pm29, Pm32, Pm34, and Pm35 originated from Aegilops (Miranda et al. 2007); Pm21 originated from Haynaldia (Cao et al. 2011); Pm40 and Pm43 originated from Elytrigia (He et al. 2009); Pm4b and Pm33 originated from T. carthlicum (Zhu et al. 2005); Pm25 originated from T. boeoticum (Shi et al. 1998); and Pm6, Pm27, and Pm37 originated from T. timopheevii (Perugini et al. 2008). All provide race-specific resistance to powdery mildew.

Wild emmer (T. turgidum var. dicoccoides, AABB, 2n = 28) is a valuable source of resistance to pathogens, and genes transferred from this species included powdery mildew resistance genes Pm16, Pm26, Pm30, Pm36, Pm41, and pm42, which were transferred to wheat chromosomes 5BS, 2BS, 5BS, 5BL, 3BL and 2BS, respectively (Reader and Miller 1991; Chen et al. 2005; Rong et al. 2000; Liu et al. 2002; Blanco et al. 2008; Li et al. 2009; Hua et al. 2009). There is also potential, largely untapped of the rich genetic resource in wild emmer, for disease resistance, pest tolerance and various abiotic stresses (Xie and Nevo 2008).

Map-based cloning of genes in wheat is hampered by the large genome size (16,000 Mb) and complexity of polyploid genomes, with about 80% of repetitive DNA sequences. To date, only two powdery mildew resistance genes have been cloned, including Pm3 allelic series (Yahiaoui et al. 2004; Srichumpa et al. 2005) and Pm21 (Cao et al. 2011). However, remarkable achievements in the area of gene identification in wheat have been made recently. A total of 1,073,845 wheat expressed sequence tags (ESTs) (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html) are available in the public database (http://www.ncbi.nlm.nih.gov/), and more than 16,000 ESTs were located in specific chromosome deletion bins by the NSF wheat EST project (Qi et al. 2004). The development of many ESTs offers opportunities for a variety of studies including development of functional molecular markers (STS and SNP), particularly gene expression, and comparative genomics research.

The high level of conserved synteny to model grass genomes like Brachypodium (The International Brachypodium Initiative 2010), rice (Rota and Sorrells 2004) and barley (Drader and Kleinhofs 2010) provides useful efforts to the identification of candidate genes for traits of interest, predicting biological function of genes, fine mapping and genes cloning, such as Vrn1 (Yan et al. 2003), Vrn2 (Yan et al. 2004), and Vrn3 (Yan et al. 2006).

The powdery mildew resistance gene PmAS846 was transferred to the long arm of chromosome 5B to common wheat lines N9134 and N9738 from wild emmer wheat. The objective of this study was to construct a saturation synteny map between wheat, Brachypodium and rice, focusing on the genomic region harboring PmAS846. The markers tightly linked to PmAS846 should be useful for marker-assisted selection (MAS) and cloning of this gene.

Materials and methods

Plant materials

Two segregating populations were developed for the mapping of PmAS846. The initial mapping population included 129 F2 plants and the derived F2:3 families from the N9738/Huixianhong cross, and the low level of polymorphism observed between the parental lines of this cross prompted the development of new mapping population. The second population included 362 F2 plants and derived F2:3 families from the cross of N9134/Shaanyou 225. N9134 is a resistant common wheat line carrying PmAS846 introgressed from wild emmer accession As846 (T. dicoccoides). In a previous work, this gene was located on chromosome 5BL by the simple sequence repeat (SSR) marker Xgwm67 with a genetic distance of 20.6 cM (Wang et al. 2007). It confers broad spectrum resistance to wheat powdery mildew. The common wheat line N9738, carrying PmAS846, was developed from the cross N9134/Zhong 4 (Wheat-Thinopyrum intermedium partial amphiploid, and highly susceptible to powdery mildew). Huixianhong and Shaanyou 225 are common wheat varieties that are highly susceptible to Bgt isolate E09. Chancellor was used as a susceptible control in the disease reaction tests. Chinese Spring (CS) and its chromosome 5B deletion lines (5BL-09, 5BL-11, 5BL-13, 5BL-14, 5BL-16 and 5BS-04, kindly provided by Drs Takashi Endo and Shuhei Nasuda, Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Japan) were used for chromosomal arm assignment and bin mapping of molecular markers.

Evaluation of powdery mildew reactions

Powdery mildew reactions of the F2 mapping population and F2:3 families (comprising 20 seedlings per family) were assessed via inoculation with Bgt isolate E09 (kindly provided by Drs Xiayu Duan and Yilin Zhou, State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China). E09 is a single-spore isolate with the following virulence response: Pm1a, Pm3b, Pm3c, Pm3e, Pm5a, Pm6, Pm7, Pm8, Pm17 and Pm19, which was maintained on Chancellor until the leaf was fully expanded by conidia. Plants were inoculated by dusting conidia from sporulating seedlings of Chancellor at the two to three leaf stages. The susceptible parents Shaanyou 225, Huixianhong and Chancellor were used as the susceptible controls, and the resistant parents N9134 and N9738 as the resistant controls in the disease reaction test. Phenotypes were recorded 12 days after inoculation when the susceptible controls were fully infected, and disease evaluations were performed according to Sheng (1988). Six infection type (IT) classes (0, 0;, 1, 2, 3, and 4) were scored, in which grade “0” represents immune reaction with no visible symptoms in plants; grade “0;” represents highly resistant reaction with hypersensitive necrotic flecks on leaves; grade “1” is highly resistant reaction with minute colonies on leaves, smaller than 1 mm in general with few conidia; grade “2” represents moderately resistant, at which leaves have moderately developed hyphae but colonies smaller than 1 mm, and some conidia; grades “3” and “4” are moderately susceptible (colonies with well-developed hyphae and abundant conidia, but colonies do not joint together) and highly susceptible (colonies with well-developed hyphae and abundant conidia, colonies are mostly joint together) types, respectively.

Genomic DNA isolation and SSR marker analysis

DNA was isolated from leaves of the entire F2 population and of the parental lines by the CTAB method (Saghai-Maroof et al. 1984). Two resistant bulks and two susceptible bulks were made by pooling equal amounts of DNA from 10 resistant or 10 susceptible F2 plants, respectively. Because PmAS846 was previously assigned to wheat chromosome 5BL, 72 SSR markers specific for wheat chromosome 5BL were screened for polymorphisms on the parents and the bulks. Polymorphic markers indicative of linkage with PmAS846 were further used to genotype the entire F2:3 mapping population to determine genetic linkage between the gene and the markers. SSR markers were selected from the primer sets GPW (http://wheat.pw.usda.gov/ggpages/SSRclub/GeneticPhysical/), GWM (Röder et al. 1998), WMC (Somers et al. 2004), BARC (Song et al. 2005) and FCP (Zhang et al. 2009). The sequences of SSR primers were obtained from the GrainGenes Database (http://wheat.pw.usda.gov/GG2/index.shtml).

PCR amplifications were performed in a S1000 Thermal Cycler (Bio-Rad, California, USA) in 10 μl volume containing 1 μl of PCR buffer (10 mM Tris–HCl, 50 mM KCl, pH 8.3), 1.5 mM of MgCl2, 0.25 U of Taq DNA polymerase, 0.2 mM of dNTPs, 0.5 μM of each primer, and 50–100 ng of template DNA. PCR conditions for different markers included a 95°C denaturing step for 3 min, followed by 35 cycles of 95°C for 45 s, 50–65°C annealing (depending on annealing temperature of the primer pairs) for 45 s, and 72°C for 45 s to 1 min (depending on PCR product sizes), and a final extension at 72°C for 10 min. The amplification products from the markers were separated in 8% polyacrylamide gels or 2% agarose gels. PCR products were visualized with silver staining or ethidium bromide and photographed.

Chromosome arm assignment and physical bin mapping

The physical locations of markers linked to the resistance gene on chromosome 5BL were determined using CS chromosome 5B deletion lines characterized by Endo and Gill (1996).

Data analysis

Chi-squared (χ2) tests were conducted to determine the goodness of fit of segregation ratios to theoretical Mendelian ratios. Linkages between markers and the resistance gene were established using JoinMap4.0 (http://www.kyazma.nl/index.php/mc.Join-Map/sc.General/), with a LOD threshold of 3.0.

Comparative mapping and marker development based on collinearity of Brachypodium and rice

Information regarding ESTs previously mapped to the deletion bin 5BL14-0.75–0.76 covering the PmAS846 interval was obtained from GrainGenes wEST-SQL resources (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi). These sequences were used for developing EST-STS markers using Primer 3 software (http://frodo.wi.mit.edu/primer3/). EST-STS primers (MAG set) assigned to chromosome 5BL (Xue et al. 2008), and EST-SSR marker BJ261635 mapped to chromosome 5BL and closely linked with Pm36 (Blanco et al. 2008) were also screened for polymorphism. The original sequence of marker MAG2498 was kindly provided by Dr. Zhengqiang Ma, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, China.

Mapped EST sequences were blasted against the Brachypodium (http://www.brachypodium.org/) and rice genomes (http://rice.plantbiology.msu.edu/) using BLASTn. Brachypodium and rice genes with the best hit (e values >1E−10 and >80% nucleotide identity) and/or within the syntenic regions were then used as queries in BLASTn searches of Triticum sequences (http://wheat.pw.usda.gov/GG2/blast.shtml). The Triticum sequences were aligned with Brachypodium and rice CDS and genomic sequences. A total of 156 conserved primer pairs were designed using Primer 3 and Conserved Primers 2.0 softwares (You et al. 2009).

PCR amplification was performed in a similar procedure to the SSR marker survey described above. For EST markers that were monomorphic between the parents on PAGE gels, single-stranded conformational polymorphism (SSCP) analyses were employed (Sunnucks et al. 2000). The PCR products were mixed with equal volume of formamide loading dye (98% formamide, 10 mM EDTA, 0.25% bromophenol blue, and 0.25% xylene cyanol), denatured at 95°C for 10 min, then placed on ice, and 5.0 μl of the mixture was loaded onto 8% non-denaturing polyacrylamide gels (37.5:1 acrylamide: bis-acrylamide). Gels were run at 4°C and 4 W for 10 h and the DNA fragments were visualized by 0.2% silver staining.

Results

Evaluation of powdery mildew reactions

When inoculated with the Bgt isolate E09, N9134 and N9738 were highly resistant (IT = 0 to 0;), “0;” is a IT, whereas Shaanyou 225 and Huixianhong were highly susceptible (IT = 4). The N9738/Huixianhong population with 129 F2 plants segregated in 94 resistant and 35 susceptible when inoculated with E09 (Table 1), indicative of a single dominant resistance gene conferring powdery mildew resistance (\( \chi_{3:1}^{2} \) = 0.313, df = 1). A total of 362 F2 plants derived from the cross N9134/Shaanyou 225 were also inoculated with E09 and showed a segregation as 266 resistant and 96 susceptible, which fit the expected 3:1 ratio for a single dominant gene (\( \chi_{3:1}^{2} \) = 0.446, df = 1). The segregation in both F2:3 families fits 1:2:1 ratios (\( \chi_{1:2:1}^{2} \) = 2.379 and 1.889, respectively, df = 2), confirming that the powdery mildew resistance in N9134 and N9738 is conferred by a single dominant gene.

Table 1 Segregation ratios of PmAS846 in the two F2 populations
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Identification and bin mapping of SSR markers

Initially, 72 wheat SSR markers on chromosome 5BL were screened for polymorphisms between N9738 and Huixianhong and between resistant and susceptible bulks. Twelve polymorphic markers, Xbarc88, Xbarc74, Xwmc75, Xwmc415, Xwmc537, Xgwm408, Xgwm67, Xgpw3035, Xgpw358, Xgpw3191, Xgpw7346 and XFCP1, were selected to genotype 129 F2 plants of N9738/Huixianhong for the construction of linkage map. Two flanking SSR markers, Xgpw7346 and XFCP1, were closely linked to the resistance locus at 1.7 and 1.3 cM, respectively. SSR markers previously mapped between two specified loci Xbarc74 and Xwmc75 (http://wheat.pw.usda.gov) were also tested for polymorphism between N9134 and Shaanyou 225. Seven markers (Xgpw7309, Xgpw3191, Xgpw7346, Xgpw7425, XFCP1, XFCP620 and XFCP394) revealed polymorphisms between two bulks and were subsequently used to genotype 362 F2 plants in N9134/Shaanyou 225 population. PmAS846 was flanked by SSR loci Xgpw7346 and XFCP1 with genetic distances of 0.8 cM proximal and 0.9 cM distal, respectively.

In order to determine on which chromosome bin the target gene resides, CS and its deletion lines of chromosome 5B were used to physically map flanking markers. Both Xgpw7346 and XFCP1 were located to the distal end of chromosome 5BL (bin 5BL14), indicating that the PmAS846 gene was located in the physical bin 5BL14 (0.75–0.76) (Fig. 1).

Fig. 1
figure 1

Amplification pattern of markers Xgpw7346 (a) and XFCP1 (b) in Chinese Spring and its 5B chromosome deletion lines. M D2000 ladder, CS Chinese Spring, CS deletion line: 5BL-09 (FL = 0.76), 5BL-11 (FL = 0.59), 5BL-13 (FL = 0.82), 5BL-14 (FL = 0.75), 5BL-16 (FL = 0.79), 5BS-04 (FL = 0.43), FL fraction length

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Bin-mapped EST marker analysis

To further delineate the genetic location of PmAS846, 21 EST-STS markers and one EST-SSR marker BJ261635 were screened. Five markers (BF482522, BF202652, BF484437, MAG2498 and BJ261635) were polymorphic between N9738 and Huixianhong (Table 2). These were subsequently mapped in the N9738/Huixianhong F2 population. The EST-SSR marker BJ261635 displayed a nearly consistency between genotype and phenotype (Fig. 2).

Table 2 Summary of Brachypodium and rice collinearity-based marker development for the PmAS846 region
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Fig. 2
figure 2

Polyacrylamide gel electrophoresis of PCR products amplified with marker BJ261635 in the F2 population of N9134/Shaanyou 225. Lanes 2 N9134; 3 Shaanyou 225, 4 resistant bulk, 5 susceptible bulk; 6–14 nine susceptible F2 plants; 15–23 nine resistant F2 plants. Arrow indicates the polymorphic amplification products. A D2000 ladder is shown in lanes 1 and 24

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High-density genetic mapping of PmAS846 based on collinearity between wheat, Brachypodium and rice

The high level of collinearity exists between wheat chromosome 5BL, Brachypodium chromosome 4 and rice chromosome 9 (The International Brachypodium Initiative 2010; Linkiewicz et al. 2004; Rota and Sorrells 2004), which may provide useful information for fine mapping of this gene. To develop additional markers, we evaluated the levels of collinearity between the PmAS846 region, Brachypodium and rice using sequences of five mapped ESTs and one SSR marker XFCP620. SSR marker XFCP620 is at the locus of gene WK35 (Faris et al. 2010), and its orthologous gene in Brachypodium and rice were Bradi4g38010 and Os09g38910, respectively. BLAST searches revealed that all the five mapped wheat sequences had significant similarity to sequences on Brachypodium chromosome 4 except MAG2498, and three of them had similarity to sequences on rice chromosome 9 based on searches of rice sequences except BF484437 and MAG2498 (Table 2). The region around the PmAS846 locus on wheat chromosome 5BL was identified to be syntenic to part of Brachypodium chromosome 4 and rice chromosome 9.

Genes located in the collinear regions of Brachypodium chromosome 4 and rice chromosome 9 were selected to develop conserved markers for saturating the PmAS846 region. Based on the identified Triticeae sequences, 156 conserved primers were designed using the Conserved Primers 2.0 and Primer 3 online. Twenty-one markers were closely linked to PmAS846 in the two mapping population. Most of these markers are co-dominant, such as RG-36976 and RG-37900 (Fig. 3). The marker RG-36976 with a NBS-LRR analog was designed to allow unequivocal distinction of homozygous and heterozygous genotypes by both agarose gel and polyacrylamide gel electrophoresis (Fig. 3). Seven closely linked markers were mapped within a genetic interval of 0.8 cM (0.5 and 0.3 cM on either side of the gene) region carrying PmAS846, and five of them, BJ261635, AL819406, CJ694617, CJ540214, and RG-37900, co-segregated with PmAS846 in the N9134/Shaanyou 225 population (Fig. 4).

Fig. 3
figure 3

PCR products of markers RG-36976 (a polyacrylamide and agarose gel electrophoresis, respectively) and RG-37900 (b polyacrylamide gel electrophoresis) amplified in the F2 population of N9134/Shaanyou 225. Lanes 2 N9134, 3 Shaanyou 225, 4 resistant bulk, 5 susceptible bulk, 6–11 six homozygous susceptible individual genotypes, 12–17 six heterozygous resistant individual genotypes, 18–23 six homozygous resistant individual. Arrow indicates the polymorphic amplification products. A D2000 ladder is shown in lanes 1 and 24

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Fig. 4
figure 4

Molecular mapping of the PmAS846 locus in the F2 populations derived from N9738/Huixianhong (b) and N9134/Shaanyou 225 (c). The genetic linkage map of the PmAS846 region corresponds to the 5BL deletion bin 5BL14-0.75–0.76 (a). Markers are indicated to the right side of the genetic map. Flanking SSR markers for PmAS846 are shown in green and connected with dashed lines

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In summary, the genetic linkage map of the PmAS846 region contained 42 molecular markers including 16 SSR markers and 26 EST-derived markers. Comparative sequence analysis revealed good levels of collinearity in the PmAS846 region, Brachypodium chromosome 4 and rice chromosome 9, but with a couple of exceptions between PmAS846 region and Brachypodium chromosome 4, and six exceptions between Brachypodium chromosome 4 and rice chromosome 9. The collinear region covered ~1.25 Mb (41.95–43.20 Mb) and ~0.99 Mb (21.39–22.38 Mb) on Brachypodium chromosome 4 and rice chromosome 9, respectively (Table 2).

Comparative mapping in the two populations revealed that BJ261635 was the proximal marker which was 1.1 cM from PmAS846 in the N9738/Huixianhong population, but co-segregated with PmAS846 in the N9134/Shaanyou 225 population. Therefore, the two flanking EST markers BJ261635 and CJ840011 are the most closely linked markers, and narrowed PmAS846 locus to a region that is collinear with 197 and 112 kb genomic regions on Brachypodium chromosome 4 (Bradi4g37680 to Bradi4g37960) and rice chromosome 9 (Os09g38520–Os09g38755), respectively.

A synthetic and orthologous gene comparison map is presented in Fig. 5; only Bradi4g36976 (chromosome 4: 42.18 Mb) and Bradi4g38170 (chromosome 4: 43.17 Mb) in the Brachypodium collinear region spanning PmAS846 are annotated as disease resistance gene located at the distal end of chromosome 4L (http://www.gramene.org/Brachypodium_distachyon/Info/Index). Two markers, RG-36976 and RG-38170, in the N9134/Shaanyou 225 population are orthologous to Bradi4g36976 and Bradi4g38170 in the collinear region of Brachypodium, and they linked with PmAS846 at 1.4 and 0.9 cM, respectively.

Fig. 5
figure 5

Comparison of the PmAS846 region on wheat chromosome 5BL (a, d) with Brachypodium distachyon chromosome 4 (b) and rice chromosome 9 (c). Markers of wheat and genes of Brachypodium or rice with collinearity are connected with dashed lines. The collinear genes are indicated to the left of Brachypodium chromosome 4 and rice chromosome 9 based on chromosome Mb positions. Collinear region comprising the genes Bradi4g37680 and Bradi4g37960, which span PmAS846, are indicated in green. Map position of Pm36 is according to Blanco et al. (2008); map position of Ml3D232 is according to Zhang et al. (2010) (color figure online)

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There are 28 gene sequences (CDS, TIGR accession numbers from Bradi4g37680 to Bradi4g37960) among the corresponding homologous region in Brachypodium that could be considered for further marker saturation. Twenty-three of these genes on Brachypodium chromosome 4 with their annotations are shown in Table 3. No significant homologies with disease resistance protein sequences were found in the collinear regions of Brachypodium (Bradi4g37680 to Bradi4g37960) and rice (Os09g38520 to Os09g38755) (http://www.gramene.org/). However, Bradi4g37710 was annotated as homing “serine/threonine phosphatase activity” and Bradi4g37900 as “leucine-rich repeat protein kinase”. Bradi4g37800 was homologous with the Arabidopsis gene AT2G03070, located on Arabidopsis chromosome 2 that is known to play a role in defense responses to fungi (http://www.arabidopsis.org/index.jsp). Ontology analysis of these genes on collinear region of Brachypodium indicated that Bradi4g37720 and Bradi4g37740, like Bradi4g37710 and Bradi4g37900, are responsive to external or biotic stimuli. These results indicated that all the genes and their homologs might play important roles in defense response processes in wheat and could be considered as potential candidate genes for PmAS846.

Table 3 Candidate genes in the Brachypodium genome bd21 8X release sequence located in the collinear region comprising the markers BJ261635 and CJ840011, which span PmAS846 in wheat N9134 and N9738
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Discussion

The powdery mildew resistance gene PmAS846 was introgressed into common wheat line N9134 from the wild emmer accession As846 and subsequently transferred to common wheat line N9738. PmAS846 provides a potent resistance that is effective against 21 Chinese Bgt isolates with different virulence patterns (our unpublished date), and it should be a valuable resource in wheat breeding programs. In the present study, a genetic linkage map for PmAS846 was developed using 42 SSR and EST-based markers and spanned a genetic distance of 50.6 and 7.2 cM in the two mapping populations, respectively. SSR markers Xgpw7346 and XFCP1 flanking PmAS846 were located in bin 5BL14-0.75–0.76, within a gene-rich region identified previously.

Many important disease resistance genes were located to wheat chromosome 5BL, including Pm36 (Blanco et al. 2008), Tsn1 (Faris et al. 2010), Ml3D232 (Zhang et al. 2010) and many others. Pm36 was transferred into durum wheat line 5BIL-29 and 5BIL-42 from wild emmer accession MG29896. Ml3D232 was transferred into the hexaploid wheat line 3D232 from wild emmer accession I222. EST marker CJ683537 (a putative NBS-LRR sequence) co-segregated with Ml3D232 is orthologous to Bradi4g36976 (Bradi4g36980, previously designated) in the collinear region of Brachypodium (Zhang et al. 2010). The EST sequences CJ683537 and BF484437 belong to a member of UniGene Ta.25929. To characterize the relationship between Ml3D232 and PmAS846, two markers BF484437 and RG-36976 were designed based on the sequences of wheat EST BF484437 and UniGene Ta.25929. Both BF484437 and RG-36976 were located on the proximal side of PmAS846 with 1.4–4.0 cM in the two genetic linkage maps of the PmAS846. EST sequences CJ832481 and BJ261635 belong to the same UniGene Ta.50830, which was homologous with the Brachypodium gene Bradi4g37680. Marker CJ832481 was located on the distal side of Ml3D232 at a genetic distance of 2.7 cM (Zhang et al. 2010). BJ261635 was the proximal marker with a 1.1 cM from PmAS846 in the N9738/Huixianhong population and co-segregated with PmAS846 in the N9134/Shaanyou 225 mapping population (Figs. 4, 5). Comparative genetic map analysis confirmed that PmAS846 was located in a more distal interval (on the distal side of Ml3D232) defined by markers CJ679871, RG-36976/BF484437/CJ683537, CK210589, CD871658 and BJ261635/CJ832481 (Zhang et al. 2010) (Fig. 5).

The EST-SSR marker BJ261635 closely linked to Pm36 was located on chromosome 5BL bin 5BL6-0.29-0.76 and on the distal side of Pm36 at 0.3–0.4 cM (Blanco et al. 2008). A combination of genetic maps (Fig. 5) indicated that Pm36 and PmAS846 are likely to either allelic or tightly linked, and an allelism test would be necessary to understand the relationships between Pm36, Ml3D232 and PmAS846.

In addition, SSR markers XFCP394 and XFCP620 flanking Tsn1 (Faris et al. 2010) were located on the distal side of PmAS846 at a 0.9 cM in the N9134/Shaanyou 225 mapping population (Figs. 4, 5). Pm36, Ml3D232, PmAS846 and Tsn1 may belong to the same gene clusters. Clusters of genes conferring resistance to wheat diseases on wheat chromosomes are not randomly distributed (Dilbirligi et al. 2004). Genes within a cluster can be allelic or closely linked, for example, the powdery mildew resistance genes at the Pm1 (Singrün et al. 2003) and Pm3 loci (Srichumpa et al. 2005).

Chromosome 5B is nearly 870 Mb (5BL, 580 Mb and 5BS, 290 Mb) and is known to contain a number of resistance genes such as Pm36 (Blanco et al. 2008), Tsn1 (Faris et al. 2010), Ml3D232 (Zhang et al. 2010) and PmAS846, Ph1 involved in homoeologous pairing (Sidhu et al. 2008), and SKr and Kr1 related to intergeneric crossability of wheat (Alfares et al. 2009). Chromosome 5B seems to have important biological functions in wheat breeding. Sequencing chromosome 5B will provide a platform for map-based cloning of interesting genes located on this chromosome (http://www.wheatgenome.org).

Wheat has an extremely large genome with more than 80% repetitive DNA sequences which make cloning of agronomically important genes very difficult (Gupta et al. 2008). However, gene-rich regions contain less repetitive DNA sequences and recombinations occur much more frequently in the gene-rich regions than gene-poor regions. The bp/cM estimates vary from 118 kb for gene-rich regions to 22 Mb for gene-poor regions (Gill et al. 1996; Gupta et al. 2008). The PmAS846 locus was delineated to a 0.8 cM interval flanked by the EST marker BG904722 on the proximal side and the EST marker CJ840011 on the distal side, close to the end of chromosome 5BL in a region known to be a recombination “hot spot”, and much recombination within the targeted interval 5BL 0.75–0.79 occurred toward the distal end (physical to genetic distance ratio ~400 kb/cM) (Faris et al. 2000). Several recombinations that occurred will give sufficient genetic resolution of the mapping populations utilized and thus an attempt at map-based cloning might be successful.

In the present study, the synteny between wheat, Brachypodium and rice was used to develop closely linked markers and to increase the density of markers around PmAS846. Within the corresponding Brachypodium genomic region (Bd4g37680–Bd4g37960), no significant homologies to a known NBS-LRR resistance gene analogy were found. In some cases, multiple rearrangements in gene order and content (non-syntenic genes) occurred, such as fungal disease resistance genes Lr10 (Feuillet et al. 2003), Lr21 (Huang et al. 2003) and Pm3 (Yahiaoui et al. 2004), the rice genome contains genes homologous to wheat genes but at non-orthologous positions. Similar situations were also found between wheat and barley and even between wheat sub genomes (Wicker et al. 2011). It seems to be consensus that macro-collinearity (collinearity on the genetic map level) is better preserved than micro-collinearity (collinearity at the sequence level).

However, even if a gene is not present on its orthologous position in Brachypodium or rice, the flanking genes are often sufficiently conserved to provide a collection of markers that can be used to saturate the target region in the other cereal genomes. For example, comparative analysis of the Tsn1 genomic region of wheat chromosome 5B with the homologous regions of rice and Brachypodium indicated a conserved level of collinearity with rice chromosome 9 and Brachypodium chromosome 4, Bradi4g38050 and Bradi4g38060 in the Brachypodium collinear region, which spans Tsn1 within a genetic interval of ~100 kb on wheat chromosome 5BL (Faris et al. 2010). Good collinearity has been shown between wheat chromosome 5BL, Brachypodium chromosome 4 and rice chromosome 9 (Faris et al. 2010; Sidhu et al. 2008; Zhang et al. 2010). In our research, the genetic linkage map of PmAS846 and Brachypodium chromosome 4 exhibited highly conserved synteny with only one exception, which seems to be better preserved than the collinearity between the wheat EST markers mapped to wheat 5BL and their putative orthologs on rice chromosome 9. The PmAS846 genomic region of wheat chromosome 5BL (BJ261635–CJ840011) also corresponds to the distal region of barley chromosome 5H (123.08-125.81 cM). As barley is more closely related to wheat than Brachypodium and rice, the recent sequencing of the barley genome (Mayer et al. 2011) should provide a new comparative genomics approach for fine mapping and cloning of genes in wheat. Markers co-segregated with PmAS846 and highly conserved synteny of this region between wheat, Brachypodium and rice, may allow map-based cloning of PmAS846. Initial attempts at chromosome walking in wheat will be performed with these orthologous gene probes that delimited physical distances of 197 and 112 kb in Brachypodium and rice, respectively.

It may be easier to use comparative maps to isolate PmAS846 from a gene-rich region on wheat chromosome 5BL using related plants with small genomes such as Brachypodium. Saturation mapping of the PmAS846 gene region with DNA markers, especially newly developed EST markers is underway. Cloning of the gene(s) will contribute to better understanding the allelic relationships, gene structure and function in this gene-rich region. In addition, most of the tightly linked or co-segregating markers for PmAS846 locus characterized in this study were inherited as co-dominant markers. They are particularly useful for MAS of PmAS846 in wheat breeding programs to quickly introgress this gene into commercial varieties or pyramid different resistance genes in a single genotype for more durable resistance.