Bmc Plant Biology Molecular Characterisation and Genetic Mapping of Candidate Genes for Qualitative Disease Resistance in Perennial Ryegrass (lolium Perenne L.)

Background: Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database.


Background
Perennial ryegrass (Lolium perenne L.) is the most widely cultivated forage, turf and amenity grass species of global temperate grazing zones. Favourable agronomic qualities include high dry matter yield, nutritive content, digestibility, palatability and the ability to recover from heavy defoliation by herbivores [1,2]. Perennial ryegrass is, however, susceptible to a number of different foliar diseases. Crown rust (Puccinia coronata f.sp. lolii) is the most widespread and damaging disease affecting ryegrasses [3][4][5][6][7]. Stem rust (P. graminis f.sp. lolii) infections are especially serious for producers of ryegrass seed [8], while grey leaf spot (Magnaporthe grisea), dollar spot (Sclerotinia homoeocarpa) and brown patch (Rhizoctonia solani) reduce turf quality [9]. The development of cultivars resistant to each of these diseases is currently recognised as an important mode of infection control.
The obligate outbreeding reproductive habit of perennial ryegrass [10] leads to high levels of genetic variation within, and to a lesser extent, between cultivars [11][12][13]. Conventional breeding for disease resistance is hence anticipated to be relatively slow for outcrossing forage species as compared to allogamous species such as cereals, because of a requirement for extensive progeny screening and phenotyping. Nonetheless, major genes and quantitative trait loci (QTLs) for disease resistance have been detected in Lolium species for resistance to crown rust [14][15][16][17][18][19][20][21], stem rust [22], bacterial wilt [23], powdery mildew [24] and grey leaf spot [25]. The extent of genetic variation within temperate Australasian crown rust pathogen populations [26] is consistent with the presence of different virulence races [27]. Identification of the molecular basis of major resistance determinants to different pathotypes will improve selection of favourable alleles during cultivar development.
Both genetic and physiological analysis has determined that hypersensitive reactions in response to fungal, viral and bacterial pathogen infections are caused by the action of genes encoding receptor proteins [28,29]. The major class of resistance (R) genes contain a highly conserved nucleotide binding site (NBS) domain adjacent to the Nterminus and a leucine-rich repeat (LRR) domain involved in the host recognition of pathogen-derived elicitors. NBS-LRRs constitute one of the largest plant gene families, accounting for c. 1% of all open reading frames (ORFs) in both rice and Arabidopsis thaliana, and are distributed non-randomly throughout the genome [30][31][32]. Clustering of R genes is known to facilitate tandem duplication of paralogous sequences and generation of new resistance specificities to counter novel avirulence determinants in evolving pathogen populations [30][31][32][33][34].
NBS domain-containing sequences have been isolated using degenerate PCR from many agronomically-important Poaceae species including cereals [33][34][35][36][37] and forage grasses [24,38,39]. In a comparison with the fullysequenced rice genome [31], only a small proportion of the total NBS domain sequences are so far likely to have been isolated from Lolium species. Multiple strategies are hence required to isolate a larger R gene sample, allowing for structural characterisation, marker development for genetic mapping, and the potential for correlation with the locations of known disease resistance loci.
Disease resistance loci of cereal species are conserved at the chromosomal and molecular level [40,41], and provide valuable template genes for a translational genomic approach to molecular marker development [42]. For example, the TaLrk10 receptor kinase gene (located at the Lr10 locus on hexaploid wheat chromosome 1AS) has been found to confer resistance to leaf rust in specific cultivars, and putative Lrk10 ortholoci are structurally conserved between Poaceae species [41,43]. The Lrk10 orthologue of cultivated oat (Avena sativa L.) exhibits 76% nucleotide similarity to the wheat gene and maps in a region of conserved synteny between the two genomes, co-locating with a large cluster of NBS-LRR genes conferring resistance to the oat form of crown rust (P. coronata f.sp. avenae) [41]. The Poaceae sub-family Pooideae includes perennial ryegrass, along with cereals of the Aveneae and Triticeae tribes [44,45], suggesting that template genes from these species are highly suitable for ortholocus isolation.
Based on studies of cereal-pathogen interactions, similar qualitative and quantitative genetic mechanisms are likely to contribute to disease resistance in perennial ryegrass. In order to test this hypothesis, a broad survey based on empirical and computational approaches was conducted to recover an enhanced proportion of perennial ryegrass NBS domain-containing sequences, as well as specific R gene ortholoci. Candidate R gene sequences (referred to as R genes throughout the text) were characterised by functional annotation, motif structure classification and phylogenetic analysis. Single nucleotide polymorphisms (SNPs) were discovered through re-sequencing of haplotypes from the parents of a two-way pseudo-testcross mapping population and validated SNPs were assigned to genetic maps. Co-location with disease resistance QTLs was demonstrated within Lolium taxa and by comparative analysis with related Poaceae species.

Bioinformatic approach to template gene selection
A proprietary resource of c. 50,000 perennial ryegrass expressed sequence tags (ESTs) [46] was integrated into the Bioinformatic Advanced Scientific Computing (BASC) system [47]. Each EST was functionally annotated using data from microarray-based transcriptomics experiments, the rice Ensembl browser, Pfam and gene ontology databases. BASC was used to search for the presence of NBS-LRR sequences. A text search with the query terms 'disease' and 'resistance' was used to identify candidates based on a wuBLASTX threshold of E = 10 -15 through known gene ontology within the genomes of closely-related cereal species (wheat, oat and barley), rice and Arabidopsis.

Primer design for candidate Lolium R genes
Locus amplification primers (LAPs) for multiple target genes were designed using standard parameters as previously described [48]. LAPs were designed from perennial ryegrass EST templates, and sequence tagged site (STS) primers derived from Italian ryegrass (L. multiflorum Lam.) NBS sequences located in GenBank [39].

Degenerate primer design
Degenerate primers (4 in sense and 12 in antisense orientation) were designed to the conserved regions (P-loop and GLPL) of cloned oat R genes [37] and were used in conjunction with published R gene-specific degenerate primers [33,34,38,49] (Additional File 1). Based on interpretation of initial amplicon complexity, specific primers were subsequently designed for SNP discovery.

Amplicon cloning and sequencing
For specific homologous and heterologous R gene-derived primers, PCR amplicons were generated using template genomic DNA from the parental genotypes of the F 1 (NA 6 × AU 6 ) mapping population [48,50]. For degenerate primers, genomic DNA from the crown rust resistant Vedette 6 genotype [14] was used as an primary template, and re-designed primer pairs were used with the F 1 (NA 6 × AU 6 ) parents. Amplicons were cloned and sequenced essentially as previously described [48], except that a total of 32 Vedette 6 clones and 12 clones from each of NA 6 and AU 6 were analysed. Trace sequence files were used as input materials into the BASC module ESTdB [47].

Classification of derived sequences
All candidate NBS-LRR (R gene) nucleotide sequences were subjected to two-way BLASTX and wuBLASTX analysis against the GenBank and the Uniprot databases, respectively. Genomic DNA sequences were translated to amino acid sequences using Transeq software. Each peptide sequence was scanned against the Pfam database [51,52] for the presence of known domains, the type, size and position of NBS domains and the number of LRR repeats. Multiple Expectation Maximisation for Motif Elicitation (MEME) [53] was used to detect conserved motifs between sequences containing NBS domains [34].

Phylogenetic analysis of R gene sequences
Preliminary alignments of predicted protein sequences was performed manually using Bioedit (version 7.0.5.3 -Ibis Biosciences, Carlsbad, CA, USA). The alignments were split into two separate datasets (for the P-Loop to GLPL region, and for the Kin-2A to GLPL region), and were realigned for phylogenetic analysis using CLUSTALX [54] with default options. Clustering of related sequences based on amino acid homology was conducted using a Neighbour Joining (NJ) algorithm and bootstrap analysis was calculated on an unrooted NJ cladogram based on 1000 iterations using CLUSTALX [55].

In vitro discovery, validation and mapping of geneassociated SNPs
PCR-amplified genomic amplicons were cloned and sequenced and DNA sequences were aligned essentially as previously described [48]. Predicted SNPs were initially validated using 10 F 1 (NA 6 × AU 6 ) individuals, and those showing Mendelian segregation were then genotyped across the full mapping panel through the single nucleotide primer extension (SNuPe) assay [48]. Integration of SNP loci into the existing F 1 (NA 6 × AU 6 ) parental genetic maps was performed as previously described [21,48,50].

Comparative genetic mapping
Comparison of chromosomal regions controlling crown rust resistance between perennial ryegrass trait-specific mapping populations was performed using data from QTL analysis of the F 1 (SB2 × TC1) mapping population [17]. The F 1 (SB2 × TC1) parental maps contained heterologous RFLP and genomic DNA-derived SSR (LPSSR) markers shared with the p150/112 and F 1 (NA 6 × AU 6 ) genetic maps, respectively [45,56]. Comparison of marker locus order between the p150/112 and F 1 (NA 6 × AU 6 ) genetic maps was performed through the presence of common LPSSR loci [50,56]. This common marker set also allowed interpolation of the position of the LpPc1 crown rust resistance locus on p150/112 LG2 [14]. Chromosomal locations of LrK10 ortholoci were compared between Lolium and Avena species using common heterol-ogous RFLP loci [58]. Further comparative genomic analysis was conducted using published genetic maps from cereal species including barley, wheat, rye and oat [33,59].

Strategies for specific R gene isolation
Three strategies (empirical approaches based on heterologous PCR and degenerate PCR, and a bioinformatic discovery method) resulted in the identification of 67 primary R gene templates for host genetic analysis (Table  1). Initial PCR amplification and resequencing using the parental genotypes of the F 1 (NA 6 × AU 6 ) mapping population allowed identification of a further 35 secondary R gene template sequences (Additional File 2). A total of 14 primer pairs amplified paralogous sequences, at a mean of 2.5 per primary template sequence, with a range from 1-12. A total of 102 distinct putative R gene sequences (corresponding to 99 NBS-containing genes and 3 receptor kinase genes) were annotated (Additional File 2) and subjected to further characterisation. Representative genomic sequence haplotypes were deposited as accessions for unrestricted access in GenBank (accession numbers FI856066-FI856167). A schematic summary of the candidate gene discovery process and further applications is depicted in Figure 1.
In the empirical approach category, translational genomics between perennial ryegrass and closely related cereal species (oat, barley and wheat) which are susceptible to other Puccinia rust pathogens (P. coronata f. sp. avenae, P. hordii, P. triticina) was used to identify R genes. Perennial ryegrass amplicons derived from oat R gene template primer pairs demonstrated high BLASTX similarity matches to their corresponding template sequences (data not shown). Primer pairs designed to the TaLrk10 template generated two 1.6 kb fragments, one of which (LpLrk10.1) displayed very high similarity scores to the putative oat ortholocus (AsPc68LrkA).
Schematic representation of empirical and bioinformatics-based discovery of perennial ryegrass R genes Figure 1 Schematic representation of empirical and bioinformatics-based discovery of perennial ryegrass R genes. Subsequent bioinformatic analysis leads to two streams of genetic analysis, including sequence characterisation, in vitro SNP discovery and large-scale genetic mapping.

LpRGcontig1
Degenerate primer pair pair Additional File 1 LpRGcontig2 Degenerate primer pair LpRGcontig3 Degenerate primer pair LpRG1NBS Degenerate primer pair LpRG2NBS Degenerate primer pair LpRG3NBS Degenerate primer pair LpRG4NBS Degenerate primer pair LpRG5NBS Degenerate primer pair LpRG6NBS Degenerate primer pair LpRG7NBS Degenerate Degenerate primer pair LpNBSC2 Degenerate primer pair LpNBSC5 Degenerate primer pair LpNBSC8 Degenerate primer pair LpNBSC15 Degenerate primer pair LpDEGVed1_d03_gp08 Degenerate primer pairs designed to oat NBS LpDEGVed2_d07_gp09 Degenerate primer pairs designed to oat NBS LpDEGVed3_a11_gp09 Degenerate primer pairs designed to oat NBS LpDEGVed4_d02_gp08 Degenerate primer pairs designed to oat NBS

LpESTa03_10rg
LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpESTa10_13rg LpEST from bioinformatic discovery LpESTb02_05rg LpEST from bioinformatic discovery LpESTb06_11rg LpEST from bioinformatic discovery LpESTc10_19rg LpEST from bioinformatic discovery LpESTd08_13rg LpEST from bioinformatic discovery LpESTe01_10rg LpEST from bioinformatic discovery LpESTe11_14rg LpEST from bioinformatic discovery LpESTf06_19rg LpEST from bioinformatic discovery LpESTf11_11rg LpEST from bioinformatic discovery The specificity of amplification using degenerate primers designed to amplify NBS domains was dependent on the proportion of deoxyinosine (I)-containing nucleotides. Those based on oat R gene templates contained a high frequency of inosines (>15%) and predominantly amplified retrotransposon-like sequences (data not shown). In contrast, combinations of largely non-degenerate primer pairs based on sequence information from multiple Poaceae species (barley, sorghum and ryegrass) (Additional File 1), successfully generated NBS domain-containing amplicons of the correct size (Additional File 3). A total of 28 distinct NBS domain-containing sequences (Tables 1, Additional File 2) were generated, several primer pairs generating multiple products (up to 7) (Additional File 3).
The text search-based computational approach identified 23 distinct perennial ryegrass ESTs with high sequence similarity to known resistance genes from closely-related species ( Database searches for previously-characterised ryegrass NBS sequences identified 51 accessions from Italian ryegrass-derived clones and a further 14 from an interspecific L. perenne × L. multiflorum hybrid (L. x. boucheanum). All 6 previously-described STS primer pairs successfully generated single amplicons of the expected size (Table 1, Additional File 2).

Molecular characterisation of perennial ryegrass R genes
From the total of 102 analysed sequences, 89 (87%) exhibited BLASTX matches at E < 10 -20 to known NBS domain-containing sequences from closely-related cereal species in both the GenBank and UniProt databases (Additional File 2). In most cases (80%), the highest matching sequence was the same for both databases. Sequence translation and subsequent Pfam analysis revealed that a substantial proportion of partial protein sequences were similar to the NBS domain (Additional File 4). A large proportion of the NBS-category sequences (55%) were within the NBS domain, while the remaining sequences either overlapped the NBS region at the N-or C-terminus, contained the LRR domain, or were located solely within the N-or C-terminal domain. A range of different R gene sub-classes containing NBS, CC-NBS, NBS-LRR, NBS-NBS-LRR, CC-NBS-LRR, CC-CC-NBS-LRR and NBS-NBS domains were detected, but no TIR-NBS containing sequences were observed. Of the different subclasses of NBS sequences, 52 contained 1-33 LRRs (modal at 3), 25 contained 1 or more CC domains, and five sequences contained the NBS-NBS domain (Additional File 4). A further three receptor kinase and NBS-LRR genes contained trans-membrane domains.
Consensuses were determined for the seven major NBS domain motifs (P-Loop, RNBS-I, Kin-2A, RNBS-II, RNBS-III, GLPL and RNBS-V) (Additional File 5) and were compared to those from closely related Poaceae species (wheat and rice) and to A. thaliana. The P-Loop, Kin-2A and GLPL motifs were most highly conserved between all species examined, while the RNBS-I and RNBS-II motifs were conserved within the Poaceae, and the Kin-2A and RNBS-II motifs were the most conserved among the CC-NBS
A total of 50 different motif signatures were identified by MEME analysis with 60 NBS domain-containing sequences at an average of 13 residues in length. The most commonly occurring signatures were components of the conserved regions such as the P-Loop, Kin-2A and GLPL motifs (Fig. 2, Additional File 6). All the distinct subclasses of NBS sequences present either completely lacked, or contained highly variable RNBS regions. Structural analysis revealed substantial diversity in motif content within the NBS domain and grouping of specific motifs into sub-classes based on shared sequence origin.

In vitro SNP discovery and genetic mapping of perennial ryegrass R genes
Sixty-five distinct R gene templates were subjected to in vitro SNP discovery through resequencing from parental genotypes of the F 1 (NA 6 × AU 6 ) mapping population. Genomic DNA of a cumulative length of c. 37 kb was analysed and a total of 819 R gene SNPs were predicted, at an overall frequency of 1 per 46 bp. A total of 11 (17%) template biparental contigs contained no SNPs, while 27 (42%) of the remaining templates contained under 10 SNPs ( Table 2). All monomorphic R gene contigs were derived from the NBS domain, apart from two encoding receptor kinase-like enzymes. SNP incidence was low within introns, due to limited representation in the sample set. SNP frequencies within parental genotypes was higher for NA 6 (38) than for AU 6 (20 R gene locus clusters were identified on a number of LGs, often in close proximity to mapped DR gene loci (represented by SNP and previously mapped EST-RFLP loci). Major clusters were identified in the lower regions of LGs 1 and 2 and the upper region of LG5 of both F 1 (NA 6 × AU 6 ) parental maps (Fig. 3, 4, 5, 6).

Comparative genetic mapping based on R gene loci
Genetic mapping facilitated map integration between trait-specific ryegrass genetic maps, and also comparative relationships with other Lolium and Poaceae taxa. Coincidences between SNP loci assigned to the F 1 (NA 6 × AU 6 ) parental maps and crown rust resistance QTLs detected in other studies were observed for LGs 1, 2, 5, and 7. Two R gene loci co-located with the crown rust resistance QTLs LpPc2 and LpPc4 in the lower region of LG1 (Fig. 8). A further two loci were assigned to the centromeric region of p150/112 LG2, 4 cM distant from the genomic DNAderived SSR locus xlpssrk02e02 which is closely associated with LpPc1. This marker locus group also co-locates with LpPc3 in the F 1 (SB2 × TC1) LG2 map, and through comparative alignment, with the hexaploid oat Pca cluster on Representation of motif patterns in the NBS domain of perennial ryegrass R gene sequences  LGB based on the position of the heterologous RFLP locus xcdo385.2 (Fig. 9). The R gene SNP locus xlprg60-216gt mapped adjacent to a previously-identified crown rust resistance QTL on AU 6 LG7, and in putative alignment with a corresponding QTL on LG7 of the Lolium interspecific hybrid ψ-F 2 (MFA × MFB) population map, but a limited number of common markers precluded further interpretation (data not shown).
Comparative genomic analysis detected conserved relationships between perennial ryegrass Lrk10 R gene SNP locus (xlprg1-369ct) and the corresponding cereal LrK10 template genes. A macrosyntenic region was identified on LG1, although low numbers of common genetic markers again limited the accuracy of extrapolation (Fig. 10). The perennial ryegrass R gene loci xlprg24-460at and xlprg54-688ag are derived from putative orthologues of the barley R genes HvS-217 and HvS-L8, respectively. Alignment of genetic maps revealed conserved syntenic locations, as well as coincidence with QTLs for leaf rust and powdery mildew resistance on barley 2H and 3H, respectively (Additional File 12, Additional File 13).

Large-scale survey of perennial ryegrass NBS domaincontaining sequences
This study describes the most comprehensive study to date of ryegrass NBS domain-containing sequences. The largest comparable surveys were of R genes from Italian ryegrass (62 sequences: [39]) and from both annual and perennial ryegrass and the corresponding interspecific hybrid (16 sequences: [38], all derived by means of degenerate primer-based amplification. In this study, 102 distinct R genes were isolated and functionally annotated. Bioinformatic analysis identified the majority of candi-date genes as members of the NBS-LRR family responsible for major gene resistance in plant species [29,[60][61][62][63][64]. A proportion of c. 20% of all perennial ryegrass R genes may be estimated to have been sampled, assuming equivalent gene content to that revealed (545 NBS sequences) by the genome-wide survey of rice [31]. It is possible, however, that major rounds of genome duplication or divergence events between species may have occurred, based on different selection pressures of surrounding pathogen populations. Such factors may influence the relative number of NBS-containing sequences in ryegrass species.

Structural classification of perennial ryegrass NBS sequences
Results from the current study suggest that only non-TIR NBS sequences are present within the Lolium genome, consistent with previous results from monocotyledonous species [33,[37][38][39]58,65]. Only degenerate primers specific to non-TIR sequences were able to amplify PCR products from perennial ryegrass genomic DNA, as observed in similar studies of sorghum [34].
Substantial variation was observed within coding regions of non-TIR NBS-LRRs, which exhibit greater sequence diversity than the TIR-NBS sub-family [66]. In this study, many R genes lacked the P-Loop region, while others contained NBS-NBS domains, duplicated CC regions or lacked CC and/or LRR domains. P-Loop, Kin-2A and GLPL motifs were conserved and similar in sequence to those of closely related Poaceae species such as wheat and rice [31,58] and the model dicotyledonous species A. thaliana [30]. Further evidence for structural gene diversity was observed within particular NBS sub-families. NBS sub-classes contained specific signature motifs between conserved regions, and in some instances, RNBS motifs Information on SNP frequencies within F 1 (NA 6 × AU 6 ) biparental contigs, preliminary validation and positions on the parental maps of the F 1 (NA 6 × AU 6 ) and p150/112 population are provided as applicable.
The key for conversion of nomenclature from R gene identifier to SNP locus identifier (rg notation) is also provided. were missing or duplicated. This suggests that the RNBS-I and RNBS-II motifs may either play a role in pathogenspecific recognition, or be less functionally significant than other, more highly conserved domains mediating resistance in plant species [30,66]. Alternatively, the presence of CC-NBS-specific motifs may suggest divergence to perform specialised functions. Variability was also observed within LRR domains, suggesting that NBS-LRRs in ryegrass are diverse in function [64,67].

Phylogenetics of Lolium NBS domain-containing sequences and relationship to genomic location and evolution
Amino acid diversity in the P-Loop-Kin-2A region may account for the major differences between TIR-NBS and CC-NBS domains. The results from this study demonstrate that TIR-NBS sequences from flax and A. thaliana group in a separate cluster, as observed in a previous phylogenetic analysis of Lolium NBS domains [38]. Further sequence analysis of a larger number of Lolium sequences in the Kin-2A-GLPL motif interval demonstrated increased sequence similarity with known TIR-NBS regions from dicotyledonous plant species, suggesting Genetic linkage maps of LGs 1-4 from the NA 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross Figure 3 Genetic linkage maps of LGs 1-4 from the NA 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross. Nomenclature for the parental maps of the F 1 (NA 6 × AU 6 ) cross is as follows: EST-RFLP markers are indicated with xlp (co-dominant Lolium perenne locus) prefixes and gene-specific abbreviations, while EST-SSR are indicated with xpps prefixes, both as described in [50]; genomic DNA-derived (LPSSR) markers are indicated as xlpssr loci using the nomenclature described in [56]. SNP loci are designated according to the nomenclature xlp-gene name abbreviation-nucleotide coordinate-SNP identity [48]. For instance, xlpchijb-240cg on NA 6 LG5 is derived from a chitinase class gene (LpCHIjb), and the SNP is a C-G transversion located at coordinate 240. DR gene SNP loci are indicated in bold red type, and corresponding RFLP loci in black bold italic type. R gene SNP loci (designated with xlprg prefixes, and numbered according to that this region may be more conserved across taxa. Consensus motif order and sequence composition indicates that the Lolium RNBS-I region may have diverged from that of dicotyledonous plants. Similar results were observed in other Poaceae species such as sorghum, for which RNBS-I consensus sequences showed significantly higher similarity to those of rice than to those of A. thaliana TIR-NBS genes [34].
Phylogenetic analysis of the P-Loop-GLPL and the Kin-2A-GLPL domains detected at least 8 NBS sub-classes, as compared to 5 separate clusters identified in a previous study [38]. Analysis of the larger number of Kin-2A-GLPL interval sequences obtained only one more cluster than for the P-Loop-GLPL interval, indicative of domain conservation. Inclusion of NBS sequences from other closely, and more distantly related, species permitted grouping of R genes and inference of possible common origins for R gene subfamilies. Sequences amplified from oat templates clus-tered together with ryegrass template-derived R genes, suggestive of a common origin. Based on known mechanisms of R gene evolution, gene duplication and divergence prior to speciation within the Pooideae sub-tribe is likely to account for the sequence similarity between ryegrass and oat genes, corresponding to putative orthologues [49,61,68].

Candidate R gene SNP discovery and genetic mapping
The SNP frequency observed within this study was marginally lower than that detected within a sub-set of 11 perennial ryegrass R genes across 20 diverse genotypes [69], but similar to that observed within DR genes [21] and a broad range of functionally-annotated candidate genes [48] in the F 1 (NA 6 × AU 6 ) mapping population. Eight R gene templates contained up to 90 SNPs per contig, possibly due to paralogous sequence alignment. Large numbers of haplotypes have been reported for other perennial ryegrass NBS-LRR genes, especially within variable LRR Genetic linkage maps of LGs 5-7 from the NA 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross Figure 4 Genetic linkage maps of LGs 5-7 from the NA 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross. Details are as described in the legend to Fig. 3 regions [69]. The data from this study suggests that allelic diversity within NBS domain is low compared to the highly variable LRR domain.
Previous studies identified significantly non-random chromosomal distributions of NBS-containing sequences [30,31]: 44 gene clusters were detected in the japonica subspecies of rice. Five major clusters containing two or more closely linked NBS-LRR genes, which frequently showed low mutual sequence similarity, were identified from only a small sub-set (26) of mapped perennial ryegrass R genes. This suggests that the gene location pattern in perennial ryegrass may be similar to that observed in other plant species. Unrelated R genes also mapped in close association with DR gene SNP and RFLP loci [21,50]. QTL based analysis and genetic mapping in wheat identified co-location of DR and R genes at qualitative disease resistance loci [70,71]. Co-location of R genes with DR genes was also observed in similar chromosomal regions (lower regions of LG1, LG2 and LG6) as disease resistance QTLs which were mapped both in F 1 (NA 6 × AU 6 ) and other trait-specific mapping populations [17,20,21].

Co-location of R gene SNP markers with disease resistance QTLs
SNP mapping of two candidate R genes in both the F 1 (NA 6 × AU 6 ) and p150/112 mapping populations has provided possible candidates for the major gene crown rust resistance QTL (LpPc1) on LG2 [14]. To determine whether R gene SNP variants are of functional significance, further experiments involving transgenic approaches, association genetic analysis or map-based cloning are required [72,73].
NBS-LRR genes loci mapping to the distal region of LG1 in the F 1 (NA 6 x AU 6 ) parental genetic maps (xlprg29-293at, xlprg30-707ag and xlprg61-23ga) are potential candidates for resistance effects to crown rust pathotypes which are yet to be identified within Australasia. Major QTLs for crown rust resistance (LpPc2 and LpPc4) have been mapped to the lower part of LG1 in 3 different perennial ryegrass trait-specific mapping populations [17][18][19] but the limited number of common markers limits accurate extrapolation between genetic maps. Two QTLs of large magnitude were identified in each of the three populations, which were screened using European crown rust Genetic linkage maps of LGs 1-4 from the AU 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross Figure 5 Genetic linkage maps of LGs 1-4 from the AU 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross. Details are as described in the legend to Fig. 3 isolates However, so far no resistance QTLs have been detected within this chromosomal region using isolates from the southern hemisphere. As both F 1 (NA 6 × AU 6 ) mapping population parental genotypes are derived from Eurasia [50], LG1-located R gene polymorphisms may confer resistance to crown rust isolates of European provenance.

Comparative genomics analysis of perennial ryegrass R genes
Comparative analysis of R gene SNP loci and corresponding ortholoci confirmed previously reported macrosyntenic relationships between perennial ryegrass and other Poaceae species [45] in nearly all instances. The sole exception was the xlprg8-271ct locus, which was assigned to LG1 despite being derived from (and highly similar to) an oat Pca template gene predicted to map to LG2. Genetic mapping of the LpLrk10 locus to LG1 suggested that the structure and chromosomal location of this gene are highly conserved throughout the Pooideae [41,43,58].
The equivalent analysis for barley R gene ortholoci provides the basis for testing R gene functionality in response to a broader range of plant diseases, requiring significant improvements of pathogen phenotyping [74] and corresponding genetic analysis [42,75].

Conclusion
This study has demonstrated that multiple approaches to R gene discovery, including the use of homologous and heterologous templates, can generate significant numbers of candidate genes for major disease resistance loci. An enhanced resource of R gene templates from perennial ryegrass has permitted evaluation of gene structural diversity and putative evolutionary origins. Efficient in vitro discovery methods allowed assignment of R gene-derived SNPs to genomic locations, revealing coincidence with pathogen resistance QTLs in ryegrasses, as well as comparative relationships with other grass and cereal species. R gene-associated markers are suitable for further evalua-Genetic linkage maps of LGs 5-7 from the AU 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross Figure 6 Genetic linkage maps of LGs 5-7 from the AU 6 parental genotype of the F 1 (NA 6 × AU 6 ) cross. Details are as described in the legend to Fig. 3 Genetic linkage maps of LGs 1,2 and 5 from the p150/112 reference population Figure 7 Genetic linkage maps of LGs 1,2 and 5 from the p150/112 reference population. Marker nomenclature for the p150/ 112 map is as follows: AFLP loci are indicated in the format exxtyyyyy (e.g., e33t50800) and heterologous RFLP loci are indicated as × plus the relevant probe name (e.g., xcdo580). Homologous RFLP loci detected by PstI genomic clones are indicated as xablpgxxx (e.g.xablpg26y). Isoenzyme and EST markers are indicated with xlp prefixes and abbrevations for gene function (e.g. acp/2 and osw). Details of SNP loci are as described in the legend to Fig. 3 Comparative mapping analysis between candidate R gene SNP loci mapped in the F 1 (NA 6 × AU 6 ) population and QTLs for crown rust resistance from other published studies Figure 8 Comparative mapping analysis between candidate R gene SNP loci mapped in the F 1 (NA 6 × AU 6 ) population and QTLs for crown rust resistance from other published studies. Alignment of NA 6 -LG1 with LpPc2 and LpPc4 on LG1 F 1 (SB2 × TC1) [17]. Marker nomenclature for the NA 6 and AU 6 maps is as described in [48,50] and the legend for Fig. 3. Marker nomenclature within the F 1 (SB2 × TC1) mapping population is described in [17] F 1 (SB2 x TC1)-LG1 Comparative mapping analysis between candidate R gene SNP loci mapped in the p150/112 population and QTLs for crown rust resistance from other published studies Figure 9 Comparative mapping analysis between candidate R gene SNP loci mapped in the p150/112 population and QTLs for crown rust resistance from other published studies. Alignment of p150/112-LG2 with the LpPc1 and LpPc3 loci on LG2 F 1 (SB2 × TC1) [17] and the Pca cluster on hexaploid oat LGB (adapted from [14]). Marker nomenclature for the p150/112 maps is as described in [48,50] and the legend for Fig. 7. The location of the LpPc1 crown rust resistance locus is as described in [14] and marker nomenclature within the F 1 (SB2 × TC1) mapping population is described in [17]. F 1 (SB2 x TC1)-LG2

Pca
Comparative mapping analysis of the perennial ryegrass LrK10 SNP locus (xlprg1-368ct) Figure 10 Comparative mapping analysis of the perennial ryegrass LrK10 SNP locus (xlprg1-368ct). Macrosynteny of putative Lrk10 ortholoci was compared in other Poaceae species through alignment with LG1 of Italian ryegrass [76], LG4_12 from hexaploid oat, 1AS from wheat, 1HS from barley and 1RS from rye [41]. Black dotted lines align common genomic DNAderived SSR markers (indicated in bold black italics) and an orange dotted line links the genetic map positions of LrK10 ortholoci.  Wheat -1AS 5.7