Introduction

Bread wheat (Triticum aestivum L., genome formula AABBDD) is a crop with one of the largest known genomes and a low gene density. Genetically, wheat is an allohexaploid species that evolved by hybridization of three closely related wild grasses: Triticum urartu (A genome), a relative of Aegilops speltoides (B genome), and Aegilops tauschii (D genome) (McFadden and Sears 1946). Among the three wheat genomes, the D-genome was the last to be acquired by polyploid wheat, about 8,000 years ago. Although the hybridization with Ae. tauschii seems to be polyphyletic, the D-genome has a low degree of polymorphism within the cultivated germplasm (reviewed by Ogbonnaya et al. 2005).

In wheat, simple sequence repeat markers (SSRs) are a powerful tool for genetic mapping (Röder et al. 1998; Pestsova et al. 2000), diversity analysis (Huang et al. 2002) and marker assisted selection (Dubcovsky 2004). Wheat SSRs have been mostly developed on a genome-wide scale (Röder et al. 1998; Gupta et al. 2002; Song et al. 2005). Consensus mapping of publicly available microsatellites has provided an overview of the saturation reached in the hexaploid wheat genome (Somers et al. 2004). However, with a total genome size of 16,000 Mbp and a content in repetitive DNA estimated to be ∼80%, there is little chance that a randomly developed marker tags a small and specific genomic region of interest. In the past, the development of microsatellite markers for specific regions of the wheat genome was not possible because of technical limitations and lack of genomic resources. More recently, some technical advances have allowed to partially overcome these problems (reviewed by Keller et al. 2005). Arrayed bacterial artificial chromosome (BAC) libraries have been developed for wheat diploid ancestors and their close relatives. Specifically, a BAC library from Ae. tauschii was developed from the accession ‘AL8/78’ which has a genome that is closely related to the wheat D-genome. The library consists of more than 300,000 BAC clones and has a 8.5×  genome coverage. Around 200,000 clones have been fingerprinted and arranged into ∼12,000 contigs (Luo et al. 2003; http://wheatdb.ucdavis.edu:8080/wheatdb/). This library provides an excellent tool to access large portions of anonymous intergenic sequences from the D-genome. In addition, pooled PCR-screenable BAC libraries have been constructed from genomic DNA of hexaploid wheat (Nilmalgoda et al. 2003).

Since the sequence of the rice genome has been released (assembly 4 of TIGR), a thorough comparative analysis of the rice and wheat genomes has demonstrated that, based on genome synteny, it is possible to predict gene order in wheat using the rice sequence as template (Sorrells et al. 2003). However, numerous exceptions to collinearity have also been found between rice and the family of the Triticeae, complicating approaches relying on comparative genomics (Bennetzen and Ramakrishna 2002; Brunner et al. 2003; Guyot et al. 2004).

The combined use of new molecular tools in wheat and rice has a high and unexploited potential for the development of microsatellite markers tagging small chromosomal regions in bread wheat. The isolation of microsatellite markers from BAC libraries for targeted genomic regions was described first in soybean by Cregan et al. (1999) and later in several other crops. Recently it was also reported in wheat by Shen et al. (2005) using an arrayed BAC library of cv. ‘Chinese Spring’, and the markers developed were tagging loci from all the three genomes. The close similarity of the bread wheat D-genome to the genome of Ae. tauschii provides a unique opportunity to use genomic information derived from Ae. tauschii for marker development in wheat. High transferability of SSRs from Ae. tauschii to wheat was demonstrated by Guyomarc’h et al. (2002).

One of the most relevant genes in wheat disease resistance breeding is Lr34, located on chromosome 7DS (Suenaga et al. 2003; Schnurbusch et al. 2004a, 2004b). Lr34 confers adult plant, durable resistance to leaf rust in bread wheat, and it was originally detected in the spring wheat material of CIMMYT (Singh 1992). In the Swiss winter wheat cv. ‘Forno’, we identified a quantitative trait locus (QTL) for leaf rust resistance, QLrP.sfr-7DS, which had a very similar mapping location and phenotype as Lr34 (Schnurbusch et al. 2004a). Therefore, although no records are available to link the pedigree of ‘Forno’ to the spring wheat germplasm at CIMMYT, QLrP.sfr-7DS and Lr34 are possibly the same locus. Lr34 is associated with the morphological character ‘leaf tip necrosis’ (LTN, Singh 1992) and the stripe rust resistance gene Yr18 (McIntosh 1992). The Lr34 locus has provided durable resistance to both leaf rust (causal fungus: Puccinia triticina Eriks.), and stripe rust (Puccinia striiformis Westend. f. sp. tritici, Ma and Singh 1996), making Lr34 a unique resource for breeding and a model for understanding the molecular basis of partial horizontal resistance. Previous work in the 7DS chromosomal region demonstrated close genetic linkage of Lr34 with SSRs GWM130, GWM295, GWM1220, and ESTs CDO475, BF473324, and BE493812 (Suenaga et al. 2003; Schnurbusch et al. 2004b; Spielmeyer et al. 2005). However, there is still no diagnostic marker available.

The aim of this study was to develop new microsatellite markers specific for the chromosomal region of Lr34, based on orthologous regions in rice and Ae. tauschii. In addition, we wanted to analyze the newly developed markers for allelic diversity in a set of wheat lines with or without Lr34 and to test their diagnostic value for marker-assisted selection.

Materials and methods

Plant material

Two Lr34 recombinant populations were used for genetic mapping: 240 ‘Arina’ × ‘Forno’ F5:7 recombinant inbred lines (Paillard et al. 2003) and a set of 600 F2 plants derived from a cross between ‘Arina’ and an ‘Arina-Lr34’ introgression line (BC2F4, with ‘Arina’ as the recurrent parent). This introgression line was verified to be homozygous for the ‘Forno’ Lr34 segment by phenotypic evaluation and with the Lr34 flanking markers Xsfr.BE493812/PstI, and Xswm5 (developed within this study).

An allele diversity study was performed on a set of lines from different breeding programs around the world for which information was available on Lr34. This set contains five ‘Chinese Spring’ chromosome-substitution lines, having the chromosome 7D of ‘Chinese Spring’ (which has Lr34, Dyck 1991) replaced by chromosome 7D of lines which do not have Lr34 (‘Cheyenne’, ‘Hope’, ‘Red Egyptian’, ‘Timstein’, and ‘Thatcher’); the line ‘Lalbahadur’ (without Lr34), and the corresponding 7D substitution line ‘Lalbahadur-Parula 7D’ (‘Parula’ has Lr34, Singh 1992); the near isogenic lines ‘Jupateco 73’, and ‘Jupateco 73R’ (with Lr34, Singh 1992); ‘Avocet’ and ‘Avocet R’ (with Lr34); ‘Thatcher Lr16’ and ‘Thatcher Lr16 + Lr34’.

Development of probes from the Lr34 orthologous region in rice

The rice BAC clones orthologous to the wheat ‘Lr34 orthologous region’ defined by the two flanking markers Xsfr.BE493812 and Xsfr.BF473324 were identified by Schnurbusch et al. (2004b). The nucleotide sequences of genes predicted by RICEGAAS (Sakata et al. 2002) in the rice BAC clones AP000399, AP003708, AP003487, AP003767, and AP003632 were compared with BLASTn to Triticeae and oat ESTs. A set of 24 ESTs of wheat, barley, and oat was identified with a similarity threshold value of 10−15 and was hybridized to wheat Southern blots to test for copy number. Sixteen probes had a low-copy number, whereas eight of them exhibited a high-copy hybridization pattern. Four of these clones were converted into low-copy probes by subcloning an intron segment of the corresponding gene by PCR. The position of the introns on the wheat ESTs was predicted aligning the coding sequence of wheat to the rice genomic DNA with the software SIM4 (http://gamay.univ-perp.fr/analyse_seq/sim4). Sequence tag site (STS) primers were designed on the EST exons flanking one of the introns as follows: STS 40 was designed on CD453029 (for. 5′-ggtgttttaagtgttttgcatgg-3′; rev. 5′-cttcaacatgactggaggacg-3′), STS 41 on BJ210832 (for. 5′-gaaagcttggaagaggcag-3′; rev. 5′-cccggttgtgccactgg-3′), STS 62 on BE516643 (for. 5′-gacatcgtgctgccggag-3′; rev. 5′-ccatccactcagagataatgacatcaa-3′), and STS 77 primers are from BJ222628 (for. 5′-ggaccgcatcttcttcagcgt-3′; rev. 5′-atcttcttcttcttcttcatctatgcacc-3′). All the probes with a low-copy hybridization pattern were first used to screen parental blots for polymorphisms, and were then hybridized to the Ae. tauschii BAC library. Parental blots included genomic DNA of the wheat cultivars ‘Forno’, ‘Arina’, ‘Chinese Spring’, and the Chinese Spring deletion line ‘7DS-4’ (Endo and Gill 1996). DNA was digested with 27 endonucleases: AluI, AvaII, DdeI, DpnII, HaeIII, MspI, TaqI, BglI, NcoI, AciI, DpnI, HindIII, MseI, SalI, NdeI, KpnI, ClaI, SacII, BamHI, DraI, EcoRI, BglII, HindIII, XbaI, EcoRV, ApaI, and SacI. Digested DNA was blotted on membranes and hybridized with 32P-labeled probes to detect polymorphisms according to standard methods (Sambrook and Russell 2001).

Bacterial artifical chromosomes library screening, sequence analysis and genetic mapping

Screening of the Ae. tauschii BAC library was performed by hybridization as described by Wicker et al. (2003) using the low-copy probes listed in Table 1. Screening of the Glenlea BAC library was done by PCR according to Nilmalgoda et al. (2003) using the PCR markers STS 77, GWM1220, and GWM295. Subcloning of BAC DNA was done as described by Stein et al. (2000). Low-pass sequencing of BAC clones was performed with ABI®3730 to identify genes and SSRs. Genes were detected with the BLASTn algorithm (Altschul et al. 1997). SSRs were identified using the SPUTNIK Perl Script (Abajian 2003). PCR primers were designed manually; primers were tested for annealing temperature and self-complementarity with the software FastPCR (Kalendar 2006). Microsatellite markers were PCR amplified from wheat genomic DNA and visualized with the LiCor® DNA Sequencer 4200. The markers polymorphic between ‘Arina’ and ‘Forno’ were mapped in the ‘Arina’ × ‘Forno’ population, consisting of 240 single seed descent lines (Paillard et al. 2003). The linkage map was constructed using MAPMAKER 3.0b for MS-DOS (Lander et al. 1987). The new DNA markers on chromosome 7DS were integrated into the previously published 7DS genetic map (Schnurbusch et al. 2004b) using the ‘try’ command and the new marker sequence was confirmed with the ‘ripple’ command. Linkage groups were drawn with the software MapChart (Voorrips 2002). Physical mapping and bin assignment was conducted using the deletion line 7DS-4 (breakpoint 0.61) derived from cv. ‘Chinese Spring’, in which 39% of the short arm of chromosome 7D is missing (Endo and Gill 1996).

Table 1 List of wheat probes detected with a BLAST search at the rice ‘Lr34 orthologous region’
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Results

Polymorphism analysis of wheat ESTs derived from the ‘Lr34 orthologous region’ of rice and identification of Aegilops tauschii BAC contigs

The two EST markers BE493812 and BF473324 flanking the QTL QLrP.sfr-7DS had been previously located on rice chromosome 6S, where they define a physical contig of ∼300 kb (Schnurbusch et al. 2004b). To increase marker density around the Lr34 locus, we identified with a BLASTn search the wheat ESTs, which are homologous to the open reading frames predicted in the rice orthologous region. The wheat ESTs were then hybridized to wheat genomic DNA to test for copy number and polymorphism, but no polymorphism was detected by any probe on the wheat D-genome in the cross ‘Arina’ × ‘Forno’. To access potentially more polymorphic intergenic regions 19 low-copy wheat ESTs and EST-derived probes (Table 1) were used to screen a BAC library of Ae. tauschii by hybridization. Seventy BAC clones were initially identified and 59 of them were found to be organized into 11 BAC contigs (Luo et al. 2003; http://wheatdb.ucdavis.edu:8080/wheatdb/). Five BAC contigs contained clusters of two to three ESTs. The corresponding genes in rice were also physically close to each other, with less than 10 kb separating them (Fig. 1). This suggests that these genes are collinear between rice and Ae. tauschii, although we cannot infer their relative physical distances in Ae. tauschii because a full BAC sequencing was not performed. One of the BAC contigs contained one cluster of genes as well as a single gene, which are conserved between rice and Ae. tauschii and are separated by a region of ∼300 kb in Ae. tauschii. In rice they are separated by 110 kb. This BAC contig of Ae. tauschii is estimated to be ∼1.2 Mbp (BAC contig containing the BAC clone HD099L21, Fig. 1).

Fig. 1
figure 1

Identification of Aegilops tauschii BAC contigs and wheat BAC clones from the targeted ‘Lr34 orthologous region’ of rice chromosome 6S. Only BACs from which new SSR were derived are shown. The SSRs detected are indicated with a white circle. Low-copy wheat, barley, and oat EST and STS probes were used to screen the BAC library of Aegilops tauschii. Probes are represented as gray boxes. Conserved gene clusters are framed by a dashed circle. The rice ‘Lr34 orthologous region’, delimited by ESTs BE493812 and BF473324, is marked with a curly bracket. TaBAC1466J2 from bread wheat cv. ‘Glenlea’ was preferred to the Aegilops tauschii homolog to maximize SSR transferability. The relative sizes of the BAC contigs of Aegilops tauschii are not drawn to scale

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For some low-copy sequences we found gene redundancy in the Ae. tauschii genome. Five probes identified two or three BAC contigs, possibly derived from different genomic regions of Ae. tauschii. To increase the probability of selecting Ae. tauschii BAC clones from the chromosomal region of interest, we selected BAC clones independently identified by at least two probes. These were BAC clones BB045B13, HD099L21, RI033N19, and HD007J19 (Fig. 1).

Selection of BAC clones from chromosome 7D of the hexaploid wheat cv. ‘Glenlea’

The BAC library of the hexaploid wheat cv. ‘Glenlea’ has a 3.5×  genome coverage, an average insert size of 85 kb and is PCR screenable (Nilmalgoda et al. 2003). Since PCR markers are more specific than restriction fragment length polymorphism (RFLP) markers, they often allow tagging only one locus of the three orthologous genomes. Therefore, the microsatellite markers GWM1220, GWM295, and the sequence tagged site marker STS 77 were used to isolate BAC clones from the BAC library of ‘Glenlea’. GWM1220 and GWM295 were described in previous studies to be linked to Lr34 (Suenaga et al. 2003; Schnurbusch et al. 2004a, 2004b; Spielmeyer et al. 2005), and we could assign them to the bin 7DS-4 by deletion mapping (data not shown). The STS 77 is an intron sequence of the EST BJ222628 (Table 1). This STS amplifies two bands of similar size, with the smaller assigned to bin 7DS-4. Only one BAC clone, TaBAC1466J2 with a size of ∼85 kb, was found to contain the lower band of STS 77. Because TaBAC1466J2 was from bread wheat, it was preferred for sequencing to the corresponding BAC clone from Ae. tauschii (Table 1). Six BAC clones were identified with GWM1220 and three with GWM295. For these two markers, the BAC clones with the largest inserts were, respectively, TaBAC940L4 and TaBAC470M18.

Identification of microsatellite sequences in BAC clones of Aegilops tauschii and bread wheat cv. ‘Glenlea’

Three BAC clones of the ‘Glenlea’ D genome (TaBAC1466J2, TaBAC470M18, and TaBAC940L4) and four from Ae. tauschii (HD007J19, RI033N19, BB045B13, and HD099L21) were sub-cloned and low-pass sequenced to identify SSRs and coding sequences. From each BAC clone a shotgun library was derived and sequenced.

Four SSRs were detected in BAC clone HD007J19, three in BAC RI033N19, only one microsatellite was found in the BACs HD099L21, BB045B13, and TaBAC1466J2 (Table 2). In TaBAC470M18 and TaBAC940L4 no additional SSRs were detected besides GWM295 and GWM1220, which had been used for screening. A BLASTn analysis of the sequences flanking the simple sequence repeats revealed that these microsatellites were not located in known coding regions or repetitive elements. The size of the repeats ranged from 18 to 134 bp (Table 3). The PCR primers were designed in the 5′ and 3′ regions flanking the repeat and were tested on genomic DNA of the lines ‘Arina’, ‘Forno’, ‘Chinese Spring’, and its deletion derivative ‘7DS-4’. SWM1, SWM5, SWM8, SWM9, and SWM10 produced bands specific for 7DS-4. SWM6 amplified two fragments, with one of them mapping to 7DS-4. SWM3, SWM4, and SWM7 were neither polymorphic, nor was it possible to physically assign them to a specific chromosome with the nullitetrasomic lines of Chinese Spring (Sears 1966). Therefore, they were not considered for further analysis. Amplification of SWM2 was successful in the Ae. tauschii accession ‘AL8/78’, but it did not amplify a fragment in any wheat line (data not shown). This SSR was probably lost during the evolution of the wheat D-genome or mutations in the primer sequences occurred.

Table 2 Sequencing and molecular characterization of BAC clones from bread wheat cv. ‘Glenlea’ and Aegilops tauschii identified with markers from the ‘Lr34 orthologous region’ of rice
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Table 3 Ten microsatellite repeats were identified in the Aegilops tauschii and wheat BAC clones of the rice ‘Lr34 orthologous region’
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Genetic mapping of microsatellites reveals a large-scale inversion between rice and wheat orthologous regions

All the newly developed microsatellites were tested for polymorphism between the parental lines for mapping in the ‘Arina’ × ‘Forno’ population (Paillard et al. 2003). SWM1, SWM5, SWM6, and SWM10 were polymorphic between the parents. SWM6 was assigned to the 7A linkage group. SWM1, SWM5 and SWM10 mapped in the 7D chromosome, closely linked to Lr34. However, in the wheat genetic map of chromosome 7D, the three SSR markers SWM1, SWM5 and SWM10 do not map inside the Lr34 flanking interval, but outside and in a position more proximal to BF473324 (Fig. 2). Based on the assumption of wheat–rice collinearity, SWM1 should have been the marker mapping closest to EST BE493812, but in wheat it is the marker, which maps most distant to it (18 cM). Similarly, SWM10 should have been the SSR with the largest distance to EST BE493812, but it is the closest (8.9 cM). SWM5 maintains its mapping position between SWM1 and SWM10, and it maps 13.6 cM proximal to BE493812. Thus, the genetic mapping in wheat suggests that the marker order in wheat is conserved, but in an inverted pattern compared to rice. This indicates the presence of a large inversion between rice and wheat ‘Lr34 orthologous regions’. This hypothesis was further supported by sequencing the wheat BAC clone TaBAC470M18, which contains the SSR GWM295. This BAC clone was found to contain a gene encoding an early noduline-like sequence (CD904551). In the wheat genetic map, GWM295 maps between SWM5 and SWM1, within the inverted region. As expected according to the observed inversion, an orthologous early noduline-like gene was located in rice between orthologous sequences to the BAC clones RI033N9 and HD007J19. The genetic map was partially verified in a population of 600 ‘Arina’ × ‘Arina-Lr34’ F2 plants (data not shown). Thus, due to the described inversion between wheat and rice, the new microsatellite markers mapped more proximal than expected assuming wheat-rice collinearity, and the microsatellite SWM10 is the closest to the Lr34 flanking interval that was defined by ESTs BE493812 and BF473324 by Schnurbusch et al. 2004b.

Fig. 2
figure 2

Genetic mapping of polymorphic microsatellites derived from BAC clones of the wheat cv. ‘Glenlea’ (TaBACs) and the wild grass Aegilops tauschii allowed to correlate the genetic map of wheat chromosome 7D with the rice physical map of chromosome 6S. The inversion detected by mapping new SSR markers is indicated by a dashed box. The BAC clones of wheat and Aegilops tauschii are not drawn to scale. Only BAC clones HB086I16 and BB045B13 belong to the same contig, however, they do not overlap. *Microsatellite GWM1220 was not polymorphic between ‘Arina’ and ‘Forno’. Its mapping position is inferred from published work (Schnurbusch et al. 2004b; Spielmeyer et al. 2005)

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Allele diversity study in wheat lines with and without Lr34

An allele diversity study was performed on a set of lines with and without Lr34 using the newly developed SWM–SSR markers, as well as the two previously published SSR markers from the Lr34 region GWM130 and GWM1220 (Suenaga et al. 2003; Schnurbusch et al. 2004b; Spielmeyer et al. 2005). The aim of this analysis was to check the degree of polymorphism at the new SSR loci in the wheat germplasm and to assess if any of the newly developed markers was diagnostic for the presence of Lr34. To assess the degree of polymorphism we used a set of lines extensively used to characterize Lr34 (Table 4, first group). All microsatellites produced an amplification product in the lines except for SWM2, which amplified a fragment only in the genomic DNA of the Ae. tauschii accession ‘AL8/78’. SWM4 and SWM7 were monomorphic, SWM3 and SWM8 produced three alleles; SWM1, SWM5, SWM6, SWM9, and SWM10 had a higher degree of polymorphism, ranging from three to nine alleles, comparable to GWM1220 and GWM130 (six and seven alleles, respectively). It is noteworthy that the SWM5 allele in the introgression line‘Arina-Lr34’ is different from both the donor parent ‘Forno’ and the recurrent parent ‘Arina’ (data not shown). It seems that in a few generations of backcrossing (BC2) and selfing (F4) this Lr34 line has developed a new allele at the Xswm5 locus. Instability observed with SWM5 might depend on the size of this repeat. The part representing only the repeat of the amplified fragment of SWM5 is 134 bp long, and is very large compared to an average SSR. It consists of (ta)32/(tg)35 in Ae.tauschii, and the repeat might be very unstable, rapidly producing new allelic variants.

Table 4 Diversity analysis of bread wheat cultivars with the SSR marker SWM10
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The microsatellite marker closest to the Lr34 confidence interval is SWM10 (Fig. 2). Interestingly, in the set of Lr34 model lines, the marker SWM10 showed an allele of the same size (allele ‘a’, 211 bp) in all the lines with Lr34, but different alleles in the lines without Lr34. Therefore, this microsatellite was tested on a broader germplasm from different wheat breeding programs to determine its diagnostic value for detection of Lr34. In Table 4, second and third section, additional elite genotypes are listed. Based on literature references it is also indicated whether they contain Lr34 or not. The lines are mostly from the CIMMYT spring wheat germplasm and from the European winter wheat germplasm. To the best of our knowledge, none of the lines of the second group has Lr34. Among this group, the only genotype having the SWM10a allele is ‘Kavkaz’. Sawhney and Sharma (1999) reported that ‘Kavkaz’ manifests good adult plant leaf rust resistance. ‘Kavkaz’ is derived from a cross with the winter wheat line ‘Bezostaja’, which has been described to have Lr34 (Winzeler et al. 2000), therefore, the line ‘Kavkaz’ might also contain Lr34, although this has not been published so far. Among the third group of lines (Table 4), all described to contain Lr34, more variability in allele size was observed. A few lines that have been described to possess Lr34 showed different bands than the ‘a’ SWM10 allele. These lines were the Canadian spring wheat cv. ‘Glenlea’ (reported to have Lr34 by Dyck et al. 1985) with the unique allele ‘e’ found in no other line, the German winter wheat line ‘Westphal 12A’ (Kolmer and Liu 2001), with allele ‘d’ and the French winter wheat line ‘Cappelle Desprez’ (McIntosh 1992), with allele ‘b’. The German winter wheat line ‘Kormoran’ shows the ‘b’ allele as ‘Cappelle Desprez’ and also contains Lr34 (McIntosh 1992). This could be explained by the presence of ‘Cappelle Desprez’ in the pedigree of ‘Kormoran’. For the Swiss winter wheat cv. ‘Titlis’ (allele ‘c’), there is no official report to support the presence of Lr34; nonetheless a strong adult leaf rust resistance and LTN lead to assume that this gene is present in ‘Titlis’ (M. Winzeler, personal communication).

Putative coding regions and repetitive DNA in bacterial artificial chromosomes clones of Aegilops tauschii and wheat cv. ‘Glenlea’

The analyzed BAC clones contained 23 putative genes, based on their homology with wheat ESTs from the TIGR database (Table 5). Nineteen sequences contained coding regions for proteins with predicted function. Two of them were homologous to the EST probes used to screen the Ae. tauschii BAC library and two others were similar to resistance genes of the nucleotide binding site-leucine rich repeat (NBS-LRR) class. In addition to these 19 sequences with predicted function, four additional sequences had significant homology to wheat ESTs, but they did not show a hit to any characterized protein. Thus, we refer to them as ‘hypothetical proteins’. The 23 predicted coding sequences were analyzed with BLASTn to identify the homologous rice genes and their localization in the rice genome. Collinearity with rice was conserved only for 11 of them, while 12 sequences, including some genes putatively encoding hypothetical proteins or resistance gene analogues of the NBS-LRR class, were not conserved in the rice orthologous region. Earlier studies have shown that in cereals, disease resistance genes of the NBS-LRR family are subject to rapid evolution and often do not maintain collinearity (Leister et al. 1998).

Table 5 A BLASTn analysis of the BAC clone sequences of wheat and Aegilops tauschii reveals the presence of a number of sequences with homology to wheat ESTs and rice genes
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Physical to genetic distances at the Lr34 locus

The three molecular markers BF473324, CDO475, and SWM10 were polymorphic on chromosome 7D in the ‘Arina’ × ‘Forno’ population, and were derived from the same Ae. tauschii physical contig, which spans ∼700 kb (Fig. 3). This allowed anchoring and orientation of a large DNA sequence of Ae. tauschii BAC clones on the wheat genetic map in chromosome 7D. In this region, the relationship between physical and genetic distances was assessed based on BAC hybridization information. The ESTs BF473324 and CDO475 were located in a ∼20 kb region (Fig. 3). SWM10 was separated from them by ∼560 kb of DNA. Thus, overall physical to genetic relationship is ∼500 kb/cM. However, this ratio varied in the region, since the 0.7 cM genetic interval defined by Xswm10 and Xcdo475 corresponded to 560 kb, while the adjacent genetic interval of 0.5 cM, defined by Xcdo475 and Xsfr.BF473324 was only ∼20 kb (Fig. 3). These two ESTs correspond to genes that in wheat have maintained a similar distance as in rice. The differences in the kb/cM ratio could possibly be explained by variations in gene content of the two intervals.

Fig. 3
figure 3

Genetic to physical distances in the Lr34 region. On the upper side a selection of BAC clones from the Aegilops tauschii BAC contig4981 is represented, according to the assembly of March 7th 2004 (http://wheatdb.ucdavis.edu:8080/wheatdb/). Below, white circles show the mapping position of three molecular markers developed from this BAC contig in the wheat 7DS chromosome. This allowed to orient the Aegilops tauschii BAC contig on the 7D chromosome, and to establish a relationship between physical and genetic distances at the Lr34 region

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Discussion

Tagging of a specific region in the D-genome of hexaploid wheat

In this work, we have tested for polymorphism a set of 24 EST markers derived from the rice ‘Lr34 orthologous region’ to saturate the Lr34 containing interval in wheat. No RFLP was detected in the D-genome between the parental lines ‘Arina’ and ‘Forno’. This outcome is consistent with the low degree of polymorphism in the D-genome of cultivated bread wheat. The SSR markers represent a good alternative for mapping because of their high level of polymorphism (Prasad et al. 2000; Singh et al. 2006). The identification of SSR markers in Ae. tauschii BAC clones allowed the enrichment of the Lr34 region on 7DS with new polymorphic microsatellite markers. Eight of nine microsatellites derived from the Ae. tauschii genome could be transferred to the D-genome of bread wheat (SWM8 was derived from the D-genome of ‘Glenlea’, SWM2 was not transferable). The Ae. tauschii BAC clones have been successfully used as a bridge to link the physical information of the rice sequence to genetic mapping in wheat. This strategy is of general utility for the development of markers for gene tagging in the D-genome of hexaploid wheat.

Low-pass sequencing of BAC clones revealed a high density of SSR motifs. In a total of 560 kb of sequence, ten SSRs were identified, giving an average of one microsatellite every 56 kb. Pestsova et al. (2000) reported microsatellite isolation from Ae. tauschii phage libraries by hybridization with the dinucleotide repeats poly(GA) and poly(GT) and found a repeat every 220 kb. In our case sequence data were available, and this allowed the detection of all the possible repeat combinations with an in silico approach. This explains the fourfold higher density of SSRs found.

Most of the developed SSRs tagged specifically the 7DS-4 bin. This was surprising as we could not exclude an occasional amplification from paralogous loci in the D-genome. In a few cases, the ESTs derived from the rice Lr34 orthologous region hybridized with BAC clones assigned by BAC fingerprinting to different BAC contigs, suggesting that they are located in paralogous regions of the D-genome. To increase the chances to tag the BACs belonging to the Lr34 region, we selected only BAC clones, which were hybridizing with probes of two or more genes closely associated in rice (Table 1). Obviously, this strategy successfully avoided the erroneous targeting of paralogous loci in the D-genome. Using BACs of diploid Ae. tauschii also reduced the targeting of orthologous loci in the A and B genomes of bread wheat, as described by Shen et al. (2005), while developing SSR markers on a genome wide scale.

Potential and limitations of the rice genome for high-resolution analysis of the wheat genome

Rice is the only grass species with a completely known genome sequence. Comparative genetic studies revealed that within the grass family there is good collinearity and, despite major rearrangements of big chromosomal segments, the order of conserved genes inside each block is frequently maintained (Bennetzen and Ma 2003). Wheat chromosome 7D is partially collinear to the rice chromosome 6. By mapping SSR and EST markers derived from the rice ‘Lr34 orthologous region’, we observed a large genomic inversion including part of the Lr34 containing interval. Small inversions affecting microcollinearity have been described earlier in Triticeae genomes (Dubcovsky et al. 2001; Isidore et al. 2005). Furthermore, in the Ae. tauschii and wheat BAC clone sequences, we identified a number of sequences with homology to ESTs that were not collinear to the targeted region of rice. These results are in agreement with earlier findings of frequent distortion of microcollinearity between rice and Triticeae genomes (Bennetzen and Ramakrishna 2002; Feuillet and Keller 2002; Akhunov et al. 2003). This mosaic conservation of microcollinearity between rice and Triticeae genomes is complicating the use of the rice model genome. However, as described below, the information from the rice genome can still provide very relevant information on candidate genes. For targeted SSR development, the application of rice genome information for the isolation of BAC clones/contigs of Ae. tauschii was extremely useful to saturate with markers a specific region of the wheat genome. A more complete physical map of wheat and/or Ae. tauschii and a better whole-genome knowledge at high resolution of rice/Triticeae genomic rearrangements would provide the necessary, improved tools for efficient work on the wheat genome.

Definition of a candidate region for Lr34

The allelic study at the Xswm10 locus indicates that this marker is genetically close to the Lr34 region. Microsatellite mapping revealed a large genomic inversion involving part of the Lr34 flanking interval. According to this information, the rice genomic region syntenic to Lr34 is now supposed to be proximal to EST marker BF473324. In this rice region, two related genes, both annotated as pectate lyase genes, were detected. Three wheat ESTs covering different parts of these rice genes were identified (data not shown). In Arabidopsis, the powdery mildew resistance gene PMR6 has a pectate lyase structure and shows good similarity at the protein level with an assembly of three wheat ESTs. At the protein level, a 73% similarity was found in a sequence of 268 amino acids. In Arabidopsis, a mutation in the gene PMR6 was described to confer horizontal resistance to powdery mildew (Vogel et al. 2002). Analysis of mutants revealed that PMR6 in Arabidopsis alters cell wall composition and has a pleiotropic effect on leaf morphology, providing a pre-haustorial kind of resistance (Vogel et al. 2002). The Lr34 locus also confers a pre-invasion resistance (Niks and Rubiales 2002), is effective against powdery mildew (Spielmeyer et al. 2005) and has pleiotropic effects on leaf morphology, e.g. it is cosegragating with LTN. Pectate lyases might play a central role in remodeling the structure of the cell wall. Therefore, they might exert a cell wall-based resistance by delaying the haustorial contact with the symplast that biotrophic plant pathogen need to establish. For this reason, we speculate that the wheat pectate lyase locus in 7DS could possibly be involved in the horizontal resistance conferred by Lr34.

Three independent sources of Lr34 ‘Frontana’, ‘Chinese Spring’, and ‘Forno’ share a common allele at the Xswm10 locus

The SWM10a allele is common to the three known independent sources of Lr34. This allele, which has a fragment size of 211 bp, is identical in the genotypes ‘Frontana’, ‘Chinese Spring’, and ‘Forno’. To our knowledge, these cultivars do not share pedigree, although genetic distances of the tested genotypes are not known. The SWM10a allele is also common to all tested spring wheat lines developed at the CIMMYT, in which Lr34 was derived from ‘Frontana’. Nonetheless, in the European winter germplasm with Lr34, more variability at the Xswm10 locus was observed. Genetic recombination, partial SSR instability or seed impurity could be involved in the observed discrepancies between presence of Lr34 and absence of the SWM10a allele in some of these genotypes. Thus, although SWM10 seems not to be completely diagnostic when using a wide germplasm, it will be an extremely useful marker to assist breeding programs for Lr34/Yr18 worldwide. Finally, the knowledge on the orientation of the SWM10 BAC contig on the 7D chromosome by genetic mapping provides the basis for chromosome walking towards Lr34.