BMC Plant Biology BioMed Central Open Access Research article Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.) Peter M Dracatos1,2,4, Noel OI Cogan1,4, Timothy I Sawbridge1,4, Anthony R Gendall2, Kevin F Smith3,4, German C Spangenberg1,4 and John W Forster*1,4 Address: 1Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, Park Drive, La Trobe University Research and Development Park, Bundoora, Victoria 3083, Australia, 2Department of Botany, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora, Victoria 3086, Australia, 3Department of Primary Industries, Biosciences Research Division, Hamilton Centre, Mount Napier Road, Hamilton, Victoria 3300, Australia and 4Molecular Plant Breeding Cooperative Research Centre, Bundoora, Victoria, Australia Email: Peter M Dracatos - p.dracatos@latrobe.edu.au; Noel OI Cogan - noel.cogan@latrobe.edu.au; Timothy I Sawbridge - tim.sawbridge@dpi.vic.gov.au; Anthony R Gendall - t.gendall@latrobe.edu.au; Kevin F Smith - kevin.f.smith@dpi.vic.gov.au; German C Spangenberg - german.spangenberg@dpi.vic.gov.au; John W Forster* - john.forster@dpi.vic.gov.au * Corresponding author Published: 19 May 2009 BMC Plant Biology 2009, 9:62 doi:10.1186/1471-2229-9-62 Received: 13 February 2009 Accepted: 19 May 2009 This article is available from: http://www.biomedcentral.com/1471-2229/9/62 © 2009 Dracatos et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Abstract 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 Results: Amplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach A total of 102 unique partial R genes were cloned, sequenced and functionally annotated Analysis of motif structure and R gene phylogeny demonstrated that Lolium R genes cluster with putative ortholoci, and evolved from common ancestral origins Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs) Conclusion: This study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-forgene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 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 [3-7] 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 [11-13] 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 [1421], 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 [30-32] 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 [30-34] http://www.biomedcentral.com/1471-2229/9/62 NBS domain-containing sequences have been isolated using degenerate PCR from many agronomically-important Poaceae species including cereals [33-37] 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 Methods 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) Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 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 NBSLRR 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] Primer design based on Pooideae R gene templates LAPs were designed based on the sequence of four oat LGB-located Pca cluster R genes [37], five barley rust resistance genes (Hvs-18, Hvs-133-2, Hvs-T65, Hvs-236 and Hvs-L6) [33]; and the third exon and 3'-terminus of the TaLrk10 extracellular domain [41] 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 F1(NA6 × AU6) mapping population [48,50] For degenerate primers, genomic DNA from the crown rust resistant Vedette6 genotype [14] was used as an primary template, and re-designed primer pairs were used with the F1(NA6 × AU6) parents Amplicons were cloned and sequenced essentially as previously described [48], except that a total of 32 Vedette6 clones and 12 clones from each of NA6 and AU6 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 http://www.biomedcentral.com/1471-2229/9/62 [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] Plant materials Perennial ryegrass genomic DNA was extracted from parents and progeny of the F1(NA6 × AU6), Vedette6 and p150/112 [45,56] mapping families using the CTAB method [57] A genotypic panel for genetic map assignment was constructed of 141 F1(NA6 × AU6) and 24 p150/ 112 F1 genotypes as previously described [21] 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 F1(NA6 × AU6) 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 F1(NA6 × AU6) 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 F1(SB2 × TC1) mapping population [17] The F1(SB2 × TC1) parental maps contained heterologous RFLP and genomic DNA-derived SSR (LPSSR) markers shared with the p150/112 and F1(NA6 × AU6) genetic maps, respectively [45,56] Comparison of marker locus order between the p150/112 and F1(NA6 × AU6) 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- Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 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] Results 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 F1(NA6 × AU6) 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 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 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) R-gene characterisation and classification into distinct classes and families Candidate resistance gene discovery Motif analysis Perennial ryegrass ESTs and GenBank-derived clones Cereal R gene ortholoci Degenerate primers oligonucleotide Amino acid alignment Bioinformatic annotation for functional role R gene sequence similarity and relationship to macrosynteny SNP discovery in F1(NA6xAU6) parental genotypes SNP validation and genetic mapping Association of SNPs with resistance loci in perennial ryegrass and cereal species Figure Schematic representation of empirical and bioinformatics-based discovery of perennial ryegrass R genes 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 Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Table 1: Classification of primary R gene templates used for host-specific genetic analysis, according to isolation strategy Perennial ryegrass unique identifier (UI) Source of primary R gene template sequence Reference LpLrk10 Wheat leaf rust receptor kinase [41] LpPcaClone1 LpPcaClone2 LpPcaClone3 LpPcaClone4 Oat NBS-LRR candidate from Pca cluster Oat NBS-LRR candidate from Pca cluster Oat NBS-LRR candidate from Pca cluster Oat NBS-LRR candidate from Pca cluster [37] LpHvClone1 LpHvClone2 LpHvClone3 LpHvClone4 LpHvClone5 Barley NBS-LRR co-locating with QTL Barley NBS-LRR co-locating with QTL Barley NBS-LRR co-locating with QTL Barley NBS-LRR co-locating with QTL Barley NBS-LRR co-locating with QTL [33] Degenerate primer pair pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pair Degenerate primer pairs designed to oat NBS Degenerate primer pairs designed to oat NBS Degenerate primer pairs designed to oat NBS Degenerate primer pairs designed to oat NBS Additional File LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery [46] Primer design based on Pooideae R gene templates Degenerate primer pair design LpRGcontig1 LpRGcontig2 LpRGcontig3 LpRG1NBS LpRG2NBS LpRG3NBS LpRG4NBS LpRG5NBS LpRG6NBS LpRG7NBS LpNBS-LRR1 LpNBS-LRR2 LpNBS-LRR3 LpNBS-LRR4 LpNBS-LRR5 LpNBS-LRR6 LpNBS-LRR7 LpNBS-LRR8 LpNBS-LRR9 LpNBSC1 LpNBSC2 LpNBSC5 LpNBSC8 LpNBSC15 LpDEGVed1_d03_gp08 LpDEGVed2_d07_gp09 LpDEGVed3_a11_gp09 LpDEGVed4_d02_gp08 Primer design for candidate Lolium R genes LpESTa03_10rg LpESTa08_14rg LpESTa10_13rg LpESTb02_05rg LpESTb06_11rg LpESTc10_19rg LpESTd08_13rg LpESTe01_10rg LpESTe11_14rg LpESTf06_19rg LpESTf11_11rg Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Table 1: Classification of primary R gene templates used for host-specific genetic analysis, according to isolation strategy (Continued) LpESTg01_20rg LpESTg04_17rg LpESTg06_13rg LpESTh04_17rg LpESTh05_28rg LpESTh07_17rg LPCL_38150 LPCL_8913 LpHvESTClone1 LpHvESTClone2 LpHvESTClone3 LpHvESTClone4 LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpEST from bioinformatic discovery LpAG205017 LpAG205018 LpAG205035 LpAG205050 LpAG205055 LpAG205063 RG sequence from Italian ryegrass RG sequence from Italian ryegrass RG sequence from Italian ryegrass RG sequence from Italian ryegrass RG sequence from Italian ryegrass RG sequence from Italian ryegrass [39] LpEST = perennial ryegrass EST; RG = resistance gene 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 (Table 1, Additional File 2) Amplification based on candidate EST primary templates was efficient, with only 13% of LAP pairs failing to generate amplicons Additional sequences were amplified from several ESTs, all were putative paralogues showing significant BLASTX similarity (E < × 10-15) to known R genes (Additional File 2) 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 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, NBSLRR, 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 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, RNBSIII, 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 Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 sequences The RNBS-III and RNBS-V motifs were highly divergent between all species 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 Phylogenetic analysis of perennial ryegrass R genes Phylogenetic analysis was performed based on two selected NBS domain regions (P-Loop-GLPL and Kin-2AGLPL) Unrelated NBS domain sequences from A thaliana, lettuce (Lactuca sativa L.), flax (Linum usitatissimum L.), tomato (Lycopersicon esculentum L.) oat, rice and barley were included for both regions, as were GenBank-derived Lolium NBS sequences A total of 38 P-Loop-GLPL sequences and 104 Kin-2A-GLPL sequences were analysed Amino acid alignment of NBS regions permitted classification into sub-families or classes A total of seven major clusters were identified for the P-Loop-GLPL region (Additional File 7, Additional File 8) Candidate sequences were clustered on the basis of similarity to putative orthologues identified from preliminary BLASTX analysis The majority were most closely related to those from other ryegrass species, although some showed highest sequence similarity to template genes from other species Sequences similar to rice R genes were also grouped with flax, lettuce and A thaliana accessions [cluster C], and a sub-set of ryegrass sequences formed two separate clusters [clusters G and H] and may hence be similar to generic R gene variants previously identified in other species, which were not included within the alignment Eight major clusters were identified for the Kin-2A-GLPL region (Additional File 9, Additional File 10) Ryegrassderived sequences were preferentially clustered with those from other Poaceae species (for instance, with oat sequences formerly used as LAP-design templates [cluster A], and with rice and barley sequences [clusters C and G, respectively]) Sequences from a number of dicotyledonous plant species were separately clustered for the PLoop-GLPL [cluster E], but co-located in several distinct clusters [cluster D and E] with ryegrass-derived sequences for the Kin-2A-GLPL region http://www.biomedcentral.com/1471-2229/9/62 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 F1(NA6 × AU6) 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 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 NA6 (38) than for AU6 (20) A further SNPs with biparental (AB × AB) segregation structures and SNPs with AA × BB structures were identified Multiple R gene SNPs from 37 (69%) of 54 SNP-containing R gene contigs were validated (Additional File 11) A total of 26 R genes were assigned to loci on the parental maps of the F1 (NA6 × AU6) mapping population (22 on all NA6 LGs [Figs 3, 4], 10 on all but LG4 for AU6 [Figs 5, 6]) SNPs in four R gene loci showed biparental segregation structures, mapping to the equivalent LG position in each parental map, and hence provide bridging markers Five loci were also mapped to equivalent positions on three p150/112 LGs A single SNP locus derived from the template sequence LpHvESTClone1.1 (xlprg50-464ca) was mapped in p150/112 but not in F1(NA6 × AU6) (Fig 7) 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 and and the upper region of LG5 of both F1 (NA6 × AU6) 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 F1(NA6 × AU6) parental maps and crown rust resistance QTLs detected in other studies were observed for LGs 1, 2, 5, and 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, 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 F1(SB2 × TC1) LG2 map, and through comparative alignment, with the hexaploid oat Pca cluster on Page of 22 (page number not for citation purposes) 30 21 21 1.2e-104 3e-102 LpPcaclone1 LpRG2NBS 2.5e-92 LpESTa10_13rg.2 3.9e-75 4e-74 LpRGcontig1 LpPcaClone3.1 LpNBS-LRR4 31 1.4e-20 Lpd07_gp09 Amino acid residues 20 27 25 30 23 48 19 13 18 60 45 1.3e-32 LpESTh05_28rg.1 29 3.9e-39 LpNBSC6 23 9.2e-43 LpHvESTClone1.1 32 1.5e-51 19 12 12 LpNBSC10 13 27 41 2.7e-54 23 LpRGcontig3 37 27 4.1e-66 LpNBSC8 90 12 20 12 20 34 49 120 46 46 46 20 49 36 19 36 24 38 150 17 17 33 17 180 36 41 17 45 28 17 17 29 35 29 34 16 20 38 38 24 33 33 35 34 48 33 29 29 14 14 32 22 33 25 7 47 10 10 28 36 31 24 24 36 36 19 35 47 47 17 17 15 11 33 12 6 29 6.9e-67 26 12 13 12 11 LpESTa03_10rg.2 23 24 40 18 18 11 49 49 4.2e 23 44 25 26 40 18 22 22 7.8e-69 27 26 11 23 20 42 8 Lpa11_gp09 27 50 18 37 42 16 16 30 9 43 43 NBS motif structurea LpNBSC2 -72 2.9e 1.6e-76 LpESTe11-14rg.3 -81 2.8e Lpd02_gp08.1 27 27 3.5e-129 LpPcaClone4.1 -100 30 2.5e AsPcaClone1 -136 3e 4.6e-161 LpESTe11-14rg.2 -170 BLASTP LpHvESTClone4.3 NBS ID 17 28 28 39 39 29 45 31 31 210 15 15 240 28 18 11 270 47 300 29 BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Figure Representation of motif patterns in the NBS domain of perennial ryegrass R gene sequences Representation of motif patterns in the NBS domain of perennial ryegrass R gene sequences Different coloured boxes and numbers indicate distinct motifs identified by the MEME program which are displayed using the MAST application (details provided in Additional File 6) (page number not for citation purposes) Page of 22 BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Table 2: Summary information for in vitro SNP discovery and genetic mapping of candidate R gene SNPs Perennial ryegrass unique identifier (UI) R gene SNP locus Identifier Number of putative SNPs/ contig size (bp) SNP frequency (per bp) Number of SNPs validated in panel of10 F1(NA6 × AU6) progeny LG location and mapped locus coordinate (cM) [F1(NA6 × AU6)] LG location and mapped locus coordinate (cM) [p150/112 population] LpLrK10.1 LpPcaClone 1.1 LpPcaClone 1.2 LpPCAClone2.1 LpPcaClone3.1 LpPcaClone3.2 LpPcaClone3.3 LpPcaClone4.1 LpHvclone1 LpHvclone2 LpHvclone3 LpRGContig1 LpRGContig2 LpRGContig3 LpRG1NBS LpRG2NBS LpRG3NBS LpRG4NBS LpRG5NBS LpRG6NBS LpRG7NBS LpNBSC5 LpESTa03_10rg.1 LpESTa08_14rg LpESTa10_13rg LpESTb06_11rg LpESTc10_19rg rg1 rg2 rg3 rg4 rg5 rg6 rg7 rg8 rg9 rg10 rg11 rg12 rg13 rg14 rg15 rg16 rg17 rg18 rg19 rg20 rg21 rg22 rg23 rg24 rg25 rg26 rg27 15/1500 3/358 0/470 0/380 1/510 0/187 0/187 1/510 0/250 0/230 2/406 4/646 2/504 0/590 18/410 17/412 14/540 4/537 20/520 25/540 15/520 3/423 23/295 24/723 5/594 10/729 9/550 107 203 N/A N/A 510 N/A N/A 510 N/A N/A N/A 162 86 N/A 30 24 36 134 26 22 35 141 77 15 119 45 96 0 0 0 0 1 0 0 3 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LG2 – 32.5 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LG2 – 62.9 N/A LG5 – 20.2 LG5 – 19.2/19.7 LpESTd08_13rg rg28 6/859 61 LpESTe11_14rg.1 LpESTe11_14rg.2 LpESTe11_14rg.3 LpESTe11_14rg.4 LpESTe11_14rg.5 LpESTe11_14rg.7 LpESTe11_14rg.8 LpESTe11_14rg.9 LpESTe11_14rg.10 LpESTe11_14rg.11 LpESTe11_14rg 12 LpESTf06_19rg.1 rg29 rg30 rg31 rg32 rg33 rg34 rg35 rg36 rg37 rg38 rg39 rg40 14/684 14/684 12/591 16/605 1/645 0/375 0/435 3/423 8/380 0/690 7/810 9/550 49 49 66 38 645 N/A N/A 141 48 N/A 116 61 2 0 0 0 LpESTf11_11rg LpESTg01_20rg LpESTg04_17rg.1 rg41 rg42 rg43 35/890 3/325 7/670 25 252 26 LpESTg06_13rg LpESTg10_13rg.1 LpESTg10_13rg.2 LpESTh04_17rg LpESTh05_28rg.1 LPCL_8913 rg44 rg45 rg46 rg47 rg48 rg49 59/880 14/540 2/540 12/604 13/580 8/600 49 143 270 108 39 2 3 LpHvESTClone1.1 LpHvESTClone1.2 rg50 rg51 90/730 2/550 73 225 NA6-LG1- 34.7 N/A N/A N/A N/A N/A N/A NA6 – LG1- 151.6 N/A N/A N/A N/A AU6 – LG2- 57.4 N/A NA6 – LG2- 172.7 N/A N/A N/A N/A N/A N/A N/A AU6 – LG1- 74.1 NA6 – LG2- 166.6 N/A AU6 – LG5 – 65.1 NA6 – LG5 – 0.0; AU6 – LG5 – 68.4 NA6 – LG5 – 10.9; AU6 – LG5 – 27.0 NA6 – LG1 – 176.1 AU6 – LG1 – 118.9 NA6 – LG2 – 161.5 N/A N/A N/A N/A N/A N/A N/A N/A NA6 – LG2 – 71.8/ 78.0 N/A AU6 – LG3 – 45.9 NA6 – LG4 – 92.8/ 94.9 NA6 – LG2 – 164.4 NA6 – LG5 – 37.1 NA6 – LG7 – AU6 – LG6 – 137.7 AU6 – LG5 -0.0 NA6 – LG6 – 134/ 134 N/A NA6 – LG5 – 105.1/124.8 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A LG2 – 32.5 N/A N/A N/A N/A N/A N/A N/A N/A N/A LG2 – 55.2 N/A Page of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Table 2: Summary information for in vitro SNP discovery and genetic mapping of candidate R gene SNPs (Continued) LpHvESTClone1.3 LpHvESTClone1.4 LpHvESTClone2.1 LpHvESTClone3.1 LpHvESTClone4.1 LpHvESTClone4.2 LpHvESTClone4.3 LpHvESTClone4.4 LpAG205017 rg52 rg53 rg54 rg55 rg56 rg57 rg58 rg59 rg60 6/556 60/690 26/670 98/1100 3/646 13/680 0/270 8/930 7/540 93 12 66 11 215 52 N/A 116 75 0 1 0 LpAG205018 LpAG205035 LpAG205050 LpAG205055 rg61 rg62 rg63 rg64 7/601 10/660 14/610 10/664 13 23 44 86 2 LpAG205063 rg65 7/602 86 N/A N/A NA6 – LG3 – 130.8 N/A N/A N/A N/A N/A NA6 – LG7 – 46.5;, AU6 – LG7 – 45.9 AU6 – LG1 – 187.4 NA6 – LG3 – 37.8 N/A NA6 – LG2 – 149.4; AU6 – LG2 – 86.9 NA6 – LG2 – 134.3 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Information on SNP frequencies within F1(NA6 × AU6) biparental contigs, preliminary validation and positions on the parental maps of the F1(NA6 × AU6) 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 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 AU6 LG7, and in putative alignment with a corresponding QTL on LG7 of the Lolium interspecific hybrid ψ-F2(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 xlprg54688ag 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) Discussion 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 [29,60-64] 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 [33,37-39,58,65] 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 Page 10 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 NA 6-LG1 0.0 34.7 56.9 66.8 71.6 76.3 79.3 81.0 85.3 89.7 93.9 96.3 97.9 100.8 101.5 103.9 108.8 111.5 114.4 115.7 117.4 120.7 122.8 128.7 132.7 133.4 135.0 138.2 141.4 142.5 143.2 143.9 151.6 155.6 156.5 158.5 159.5 160.5 162.5 164.7 176.1 176.4 176.7 177.4 xlpap2b.2 xlprg1-369ct xlpesi3f xlpesi3e.1 xlplt16ab xlpssa.2 xlplt16aa xlpa05_06Ws292 xlptrx-649ag xlpssrk12d11-038cg xlpbcd762-274at xlpcdo1173-212ac xlpzta-299ct xlpssrk10f08 xlpsalta.2 xpps0136c xpps0094a xpps0305a xlpcadc-561.45tg xlppsr168-699ag xlpcdo98-596ac xlpf5h.2 xlpssrk07f07-103at xlpcadc xlpssrk07f07 xlpssrk15h05 xpps0066b xlpssrk08a06.2 xlpssrh12a04 xlpssrh11g05 xlppif xlpssrh12g03 xlpthbna-317ag xlposeindel xlpwalie xlpwalic-170ag xlpchihs.1 xlp4cljb xlpmtd xlprg8-271ct xlpssrk14c08 xlpwalic xlpssrk10g04 xpps0381a xpps0174a xpps0055a xpps0231a xpps0038b xlprg29-293ct xlpnox-1131at xlpnox-735ag xpps0211b xpps0114a http://www.biomedcentral.com/1471-2229/9/62 NA 6-LG2 NA 6-LG3 0.0 8.8 16.4 21.1 36.0 41.4 42.5 49.5 66.1 71.8 78.0 85.1 85.8 86.5 90.2 91.6 93.5 94.9 97.4 99.3 106.2 112.2 117.2 118.7 128.1 131.3 134.3 139.8 143.2 145.2 149.4 151.5 157.8 161.5 164.4 166.6 172.7 185.6 189.3 191.0 196.4 xpps0154a xpps0259c xlppera-1041ag xlpper1 xlplt16ba xlpwalib xlpwalih xlppkabab xpps0122c xlprg40-31cgg xlprg40-284ag xpps0410b xpps0223b xpps0113b xlpssrk14b01.2 xlpssrk09g05.2 xlpssrhxx050 xlpssrk03b03.2 xlpssrk05h02.2 xlpssrk09f06.1 xlphish3-282cg xlphish3 xpps0153a xpps0037c xpps0328a xlpssrk12e03 xlprg65-202gt xlpera xlpera-376ct xpps0080a xlprg64-81at xpps0400a xpps0439a xlprg31-490ct xlprg44-514ct xlprg24-345ct xlprg15-277gt xlptc101821-122ct xlptc116908-050ct xlptc89057-116ct xlptc32601-503ac 0.0 2.5 10.6 21.6 24.8 28.6 31.4 36.2 37.8 40.3 41.0 41.7 42.6 44.4 47.1 51.1 58.5 66.8 68.4 73.0 78.4 84.1 96.4 98.5 104.7 106.3 118.4 121.5 123.5 125.5 129.9 130.8 xlpb07_06Ws249 xlpph xlpssrk03g05 xlpmtn xlpmtc.1 xlpmtl.2 xlpmtj.1 xpps0007b xlpssrhxx242 xlpssrk08b01.1 xlprg62-159ag xlpzta xpps0177c xpps0373a xpps0051a xlpcadd xlpssrk09f08 xlpssrk09g05.1 xlpssrk12h01.3 xlpssrh02d12 xpps0039b xpps0145b xlpc4h.1 xlpcysme xlpplb xpps0353b xpps0213b xpps0164a xpps0375a xlpmads1 xlphak1 xlphak1-160cg xlpnvg xlpcwnv xlpf5h.1 xlpnvc xpps0322b xlpssa.1 xlprg54-688ag NA 6-LG4 0.0 3.3 6.5 15.8 30.3 34.0 40.5 44.7 47.9 50.4 52.3 53.7 54.4 55.1 56.4 59.6 62.8 68.0 70.6 72.7 75.5 78.3 81.9 84.9 92.8 94.9 98.8 106.3 xpps0006d xlpssrh03a08.2 xlpcell xpps0150a xlpoxo-123cg xlpcluster404 xlpssrk15f05.2 xpps0146b xpps0423a xpps0201b xpps0205b xlpssrk05a11.1 xlpssrk08b11.1 xlpasra2 xlpasra2-132ag xlpa22-201ct xlpssrk01g06 xlphaka xpps0018a xlpssrk03c05 xlpchie xpps0439d xpps0433b xpps0202a xlpzba xlpssc xlpa22c xlpkabaa-858.34ct xlp4clja xlp4clja-323ag xlpomt3.1 xlprg43-403ct xlprg43-271ct xlpssrk07c11 xlpcat-561cg xlpffta.1 Figure Genetic linkage maps of LGs 1–4 from the NA6 parental genotype of the F1(NA6 × AU6) cross Genetic linkage maps of LGs 1–4 from the NA6 parental genotype of the F1(NA6 × AU6) cross Nomenclature for the parental maps of the F1(NA6 × AU6) 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 NA6 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 Table 2), are indicated in bold blue type Auxiliary DR and R gene loci mapped using JOINMAP 3.0, but not MAPMAKER 3.0, are interpolated between flanking markers to provide approximate genetic map locations 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 Page 11 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 NA 6-LG6 NA 6-LG7 NA 6-LG5 0.0 10.9 37.1 38.5 39.2 42.7 63.9 65.7 67.0 69.9 72.2 81.3 105.1 124.8 125.6 141.8 144.5 145.9 xlprg27-743ct xlprg27-912ag xlprg28-509ct xlprg45-196ct xlpcena xlpchijb-240cg xlpdefc xlpdeff xlpes3g xlpes3h xlphbb-210.230tg xlpssrk03b03.1 xlpssrk11g10 xlpssrk14c12 xlptla xpps0032b xlprg51-464ac xpps0056a xpps0111b xpps0149a xpps0199d xpps0397c 0.0 5.3 13.8 20.8 22.1 22.7 25.8 28.1 29.3 32.0 33.3 35.8 40.4 40.6 44.4 50.5 53.2 57.1 66.3 69.5 77.3 80.2 84.2 86.8 87.5 91.6 94.5 99.8 103.8 108.8 111.7 114.2 134.0 xlpsalta.1 xpps0463b xlpssrh02h05 xpps0013a xpps0432b xpps0210b xpps0098d xlpomt2 xlphbd.2 xlpspsf xlpcta-186ct xlp4cl3-1643.201ct xlpssrk05h01 xlpssrk01c04 xlpdhna.2 xlpdhna.1 xlpctaa xlpssrk11g12 xlphba.1 xlpdefa xpps0374c xpps0031b xlpdefa-233ct xpps0022a xpps0450a xlpssrk12h01.5 xlpssrk11c07 xpps0019b xlpssrk10b07 xlpssrh09e12.1 xlpcha xlpunk1-278ca xlpccra xlpdfrb xlpssrh02e01 xlpglr-1435ct xpps0299c xlprg49-105ca xlprg49-275ct 0.0 1.1 2.2 16.6 19.4 22.1 26.2 28.9 31.6 34.3 37.0 39.4 43.1 44.1 46.1 46.5 46.9 47.7 50.0 53.4 55.1 58.8 60.2 68.8 72.0 75.8 81.0 87.9 91.1 104.3 xlprg46-86gt xlpccoaomt1 xlpccoaomt1-100tg xlphba.2 xlpca xlpcana-299ca xlpsucsyn xpps0473a xlpcadlke04-98ct xlpcadlke11-86tg xlpleaa-164ct xlpssrk14b01.1 xpps0282b xpps0049b xpps0425b xpps0441a xpps0376c xlprg60-216gt xpps0131b xpps0065b xpps0466c xpps0294b xlpccha-284ga xlpznfcon3-489ct xpps0429a xpps0447b xlpssrh03a08.3 xlpssrh08h05.2 xlpa22a.1 xlpa22a.2 xlpthc.1 xlpthc.2 xlpmads4.2 Figure Genetic linkage maps of LGs 5–7 from the NA6 parental genotype of the F1(NA6 × AU6) cross Genetic linkage maps of LGs 5–7 from the NA6 parental genotype of the F1(NA6 × AU6) cross Details are as described in the legend to Fig 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-2AGLPL domains detected at least NBS sub-classes, as compared to 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 F1(NA6 × AU6) 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 Page 12 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 AU 6-LG1 0.0 4.9 37.2 63.1 70.4 71.8 74.1 74.5 74.9 75.7 76.5 77.8 78.4 78.6 78.8 79.0 79.1 79.7 80.3 82.0 84.0 88.1 90.2 91.0 95.1 99.7 103.1 105.9 108.7 110.5 114.3 118.9 144.0 153.2 187.4 xlpap2b.1 xlpap2b.2 xlpap2-305ta xpps0136a xlpesi3 xlpssrk10f08 xlprg23-177ag xlprg23-337ct xlpssrk03a02-156ct xlplt16aa xlpssrk15h05-027ct xlpssrk15h05 xlpssrk08b01.2 xlpsalta.1 xlplt16ab xlpesi3e xlpssrk08b11.2 xlp4cljb xlppsr168-388at xlpssrk12d11-038cg xpps0401a xpps0094b xlptrx-540ct xpps0255y xlposeb-896ct xlpssrh09e12.3 xlpssrk07f07 xlposeaindel xlpmtd xlppif.2 xlpssrh12g03 xlpssrh12a04 xlpthb xlpssrk12h01.2 xlpssrh09e12.2 xpps0038a xpps0114b xpps0286a xlprg30-707ag xpps0411a xlppcsa xlprg61-23ag AU 6-LG2 0.0 15.6 20.9 21.7 23.5 26.8 30.4 35.2 44.5 45.9 46.6 48.0 48.7 49.3 50.1 52.2 55.0 57.4 61.8 74.1 83.5 86.9 91.0 99.8 103.6 107.2 108.6 111.1 113.6 120.4 AU 6-LG3 xpps0121b xpps0259b 0.0 xlpssrk02c09.2 1.2 xlpssrh05f02 6.9 xlpap2b.3 9.9 xpps0218a 14.0 xlpper1.2 24.1 xpps0220b 45.9 xpps0122a 67.8 xpps0153b 72.5 xpps0223c 73.2 xpps0333a 81.2 xpps0113c 82.7 xpps0037b 88.3 xpps0410a 91.3 xlpssrk02e02 xlpssrk09f06.1 93.5 xlppkabab 94.2 xlprg13-380ag 94.8 xlpssrk02d08.2 95.4 xlphst3 96.4 xlpwesr5a 97.4 xlprg64-81at xlpera 101.4 xpps0080b 104.2 xlptc32601-514gt 109.1 xlptc101821-ct 120.3 xlptc116908-061ag 123.0 xlptc89057-072ct 126.2 xlphcd266-096cg 151.7 xpps0172b xpps0347a AU 6-LG4 xlpsfta-404ct xlpsfta.1 xlpsfta.2 xlpssrk09f06.96 xpps0198a xlpc4h.2 xlpmtn xlpmtg xlprg42-331ag xlpssrh06h02 xpps0439b xlpssrh03h02 xlpssrh02f02 xlpmtc xlpmtl.1 xlpmtl.2 xlpssrk14b01.3 xlpmtc-114.11ag xlpssrhxx242 xpps0007a xpps0373b xlpssrk09f08 xlpssrh02c11 xlpssrk14f12 xlpssrh02d12 xlpssrk15f05.1 xlpwrky14-166ga xpps0164c xlphak1f80r xlphak1 xlpssrk07h08 xlpcwinv1 xlpinvg 0.0 xpps0276a 9.1 xpps0006a 22.4 25.8 xlpcell xlpoxo-123ga 46.3 47.9 49.2 60.8 62.6 73.0 80.0 81.7 83.8 85.0 91.1 97.7 100.7 108.4 120.4 xpps0423c xpps0128b xpps0205c xpps0201c xlp4clja xlpaldpa xlpzba xlp4clja-277 xpps0433a xpps0433c xpps0003a xlpchie xpps0040b xlpffta.1 xpps0453c xlptht xlpssrk09f06.120 xpps0106a 157.4 xlpssrk02d08.b Figure Genetic linkage maps of LGs 1–4 from the AU6 parental genotype of the F1(NA6 × AU6) cross Genetic linkage maps of LGs 1–4 from the AU6 parental genotype of the F1(NA6 × AU6) cross Details are as described in the legend to Fig 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 F1(NA6 × AU6) 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 F1(NA6 × AU6) 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 F1(NA6x AU6) parental genetic maps (xlprg29293at, 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 different perennial ryegrass trait-specific mapping populations [17-19] 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 Page 13 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 AU6-LG5 AU6-LG7 AU6-LG6 0.0 7.8 12.2 13.2 27.0 57.7 63.4 64.2 65.1 68.4 71.4 72.2 72.8 74.7 77.5 79.4 82.1 83.8 85.5 89.5 94.2 99.1 102.3 106.7 108.9 113.9 115.3 121.4 144.3 152.1 xlprg48-200ct xpps0111a xlphbb-120ca xlphbb xlpinva xlprg28-340cg xlpssrh10g02 xlpssrh07g05 xlptlb xlprg26-298gt xlprg27-743ct xlprg27-912ag xlpssrk02c09.1 xlpssrk14c12 xlpssrk05a11.2 xpps0289b xlpssrh01h01 xpps0195b xpps0273a xlpssrk11g10 xlpdefc xlppih xpps0169a xpps0074a xlpssrk03b03.1 xlpmads3.1 xpps0036b xlpsaltb xlpdeff xlpgluck-394ct xlpgluck xpps0397a xlpesi3b xlpesi3g 0.0 8.8 14.2 17.2 19.5 21.5 24.7 25.5 26.6 28.3 29.9 32.8 37.6 42.3 43.0 44.0 46.0 47.5 48.3 51.8 55.5 56.7 63.3 65.7 78.9 86.5 92.6 100.4 108.1 137.7 xlpssrk01c04 xpps0098a xpps0020b xpps0210a xlpssrh08h05.1 xlpomt2 xlpssrk05h01 xlphbd xlpspsf xlpcwinv-136tg xlpinva-304ga xlpcwinv2 xlppalb xlpssrh02h05.1 xlpdefa xlpssrk11g12 xlpssrh02h05 xlpssrk13h08 xlpssrh05g07 xpps0192a xpps0374a xpps0022b xpps0310a xpps0450b xlpssrk09c10 xpps0241a xlpffta.2 xlpssrk10b07 xpps0019a xpps0187a xlpcat-353gt xlpdfrb xpps0299a xlpccra xlprg47-625gt 0.0 10.8 15.2 19.3 21.5 22.6 26.3 33.5 34.5 35.8 37.8 39.7 44.1 45.9 48.7 49.5 50.5 51.5 53.1 55.8 60.5 61.2 69.7 77.0 84.0 90.4 105.7 xlpzbb xlpssrh03a08.2 xpps0312a xlpssrk14f07 xlpa22a.2 xlpthc-148at xlpssrk12h01.4 xlpsucsyn xlpcia xlpcia-101at xpps0411c xlpssrk08a06.1 xlpleaa xpps0049c xlprg60-216gt xpps0376a xpps0441b xpps0411d xpps0466a xlpssrk05b11 xlpccha-326cg xlpomt3 xpps0438b xpps0099b xpps0424b xlpcadlike05-27.3ca xlpccha xlpcysa xlpthc.2 xlpthc.1 xlpccrb Figure Genetic linkage maps of LGs 5–7 from the AU6 parental genotype of the F1(NA6 × AU6) cross Genetic linkage maps of LGs 5–7 from the AU6 parental genotype of the F1(NA6 × AU6) cross Details are as described in the legend to Fig isolates However, so far no resistance QTLs have been detected within this chromosomal region using isolates from the southern hemisphere As both F1(NA6 × AU6) 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- Page 14 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 p150/112 - LG2 p150/112 - LG1 0.0 8.9 12.7 22.6 25.6 28.5 29.2 29.9 30.6 31.3 32.3 33.3 36.2 41.8 45.0 56.6 89.7 93.5 122.6 152.1 e33t50800 xc1239Aa e41t47500 e33t62180 xcdo580 xlpssrk10f08 xlpssrhxx238 xlpssrk14c04 xlpssrk15h05 xlpssrk09g05 xlpssrk03a02 xlpssrh02h04 xcdo98 xlpssrk07f07 xlpssrk12d11 xlpssrh09e12.2 xlpssrk14d02 xlpssrh07g03 xlpssrh12g03 xpsr601 xcdo105a xbcd1072a xlpssrk10g04 xbcd738 xpsr162 e41t47180 e33t50175 xcdo202 e41t59188 xlpnox-1131at e33t61133 0.0 13.5 15.9 22.9 26.4 27.9 29.4 32.2 32.5 35.3 36.0 36.7 38.2 39.7 41.2 42.7 44.4 49.8 55.2 58.7 62.9 77.9 86.4 88.1 96.3 102.0 111.1 114.7 116.5 129.3 xlpssrh03a08 xcdo38.1 e35t59220 e33t62225 e41t50231 xcdo405 xlpssrh02d10.1 xlpssrh09e12.1 e33t62515 e41t47225 xlprg13-380ag xlprg40-31cg xlpssrh03f03 xlpssrk13c10 xlpssrk14b06 xlpssrh01a07 xcdo385.2 xlpssrk09f06.1 xlpssrk02e02 e35t59112 e33t50133 e33t62460 xlpssrk12e03 xpsr901 e36t48595 e33t62113 e35t59575 e41t50240 xcdo1417 xlprg50-464ca xlpssrh08h05.3 xlprg24-460at xc600a e33t62620 xpsr540b e40t50334 xlpssrk08f05 xlpssrk12e06 xlpssrhxx285 xcdo36 e40t49173 xc472 xr738 xc556 xc847 p150/112 - LG5 0.0 13.1 15.4 18.7 19.2 19.7 20.2 20.7 23.5 28.9 30.6 33.8 37.3 43.6 47.1 49.6 52.2 63.1 74.0 75.7 82.7 86.0 101.0 e36t50375 xlpssrk03f09 xlpssrh11g05 xlpssrh02e12 e41t47750 xlprg27-912ag xlprg27-743ag xlprg26-298gt xlpssrk09c10 e33t50112 e41t50590 xlpssrh10g02 e41t47198 xlpssrh07g05 xlpssrk14c12 xlpssrk05h02 e41t47445 xlpssrk15a07 Xablpg26x xcdo412 orsb xlpssrk03b03 e38t50311 xr1751 e33t62210 xlpgluck-394ct e38t50189 e33t50147 e33t62101 xr2710 e41t50200 e40t50268 xcdo400 xrz404 Figure linkage maps of LGs 1,2 and from the p150/112 reference population Genetic Genetic linkage maps of LGs 1,2 and 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 Page 15 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 NA - LG1 0.0 34.7 56.9 66.8 71.6 76.3 79.3 81.0 85.3 xlprg1-369ct xlpesi3f xlpesi3e.1 xlplt16ab xlpssa.2 xlplt16aa xlpa05_071Qs292 xlptrx-649ag xlpssrk12d11-038cg xlpbcd762-274at xlpcdo1173-212ac xlpzta-299ct xlpssrk10f08 xlpsalta.2 xpps0136c xpps0094a xpps0305a xlpcadc-561.45tg xlppsr168-699ag xlpcdo98-596ac xlpf5h.2 xlpssrk07f07-103at xlpcadc xlpssrk07f07 xlpssrk15h05 xpps0066b xlpssrk08a06.2 xlpssrh12a04 xlpssrh11g05 xlppif xlpssrh12g03 xlpthbna-317ag xlposeindel xlpwalie xlpwalic-170ag xlpchihs.1 xlp4cljb xlpmtd xlprg8-271ct 0.0 PC400-014 5.3 LPSSRK03A02_b 13.7 14.4 19.9 23.9 27.0 29.9 31.2 32.7 33.7 35.5 36.1 36.3 36.9 41.0 44.4 50.6 51.8 54.0 54.3 59.7 61.7 67.5 72.6 78.8 0SE_b PC407-045 Xrgc488_b OSE_x PC400-149 LPSSRK15H05 LPSSRK10F08 LPSSRK12D11_a rye023_c PC026-03 rye023_a PC400-032 LPSSRK03A02_a LPSSRK07F07_b LPSSRK10F08_g PC400-091 LPSSRK10G04 PC008-076 PC168-023 PC026-32 PC008-085 PC026-34 PC026-R2 PC106-R2 87.6 PC168-R1 LpPc4 xlpssrk14c08 xlpwalic xlpssrk10g04 xpps0381a xpps0174a xpps0055a xpps0231a xpps0038b F1(SB2 x TC1)- LG1 LpPc2 89.7 93.9 96.3 97.9 100.8 101.5 103.9 108.8 111.5 114.4 115.7 117.4 120.7 122.8 128.7 132.7 133.4 135.0 138.2 141.4 142.5 143.2 143.9 151.6 155.6 156.5 158.5 159.5 160.5 162.5 164.7 176.1 176.4 176.7 177.4 xlpap2b.2 xlprg29-293ct xlpnox-1131at xlpnox-735ag xpps0211b xpps0114a Figure crown rust resistance from other published studies gene SNP loci mapped in the F1(NA6 × AU6) population and QTLs for Comparative mapping analysis between candidate R Comparative mapping analysis between candidate R gene SNP loci mapped in the F1(NA6 × AU6) population and QTLs for crown rust resistance from other published studies Alignment of NA6-LG1 with LpPc2 and LpPc4 on LG1 F1(SB2 × TC1) [17] Marker nomenclature for the NA6 and AU6 maps is as described in [48,50] and the legend for Fig Marker nomenclature within the F1(SB2 × TC1) mapping population is described in [17] Page 16 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 p150/112 - LG2 0.0 13.5 15.9 22.9 26.4 27.9 29.4 32.2 32.5 36.0 36.7 55.2 58.7 62.9 77.9 86.4 88.1 96.3 102.0 111.1 114.7 116.5 129.3 0.0 10.8 14.2 20.4 22.9 24.6 28.4 30.4 31.9 33.0 34.6 36.5 40.7 43.6 44.9 46.6 50.7 51.3 52.2 54.1 56.5 57.8 59.5 61.7 62.0 67.1 68.0 68.1 70.0 70.5 75.4 81.7 84.9 88.8 PC407-006 PC400-113 rye024_c PC407-055PC400-137 Xcdo385 LPSSRK14B06 PC026-R4 PC106-022 PC026-R3 PC078-121 PC026-39 rye024 Xcsu6 PC008-028 PC001-032 PC400-075 PC400-140 PC001-079 PC078-057 PC400-036 LPSSRK09G12a Xcdo456 PC106-106 PC008-088 PC168-051 PC008-050 PC400-077 PC078-075 rye022 PC400-022 PC078-099 Xbcd135 PC008-014 LPSSRK08F05_c Pca LpPc3 38.2 39.7 41.2 42.7 44.4 49.8 F1(SB2 x TC1)- LG2 LpPc1 35.3 xlpssrh03a08 xcdo38.1 e35t59220 e33t62225 e41t50231 xcdo405 xlpssrh02d10.1 xlpssrh09e12.1 e33t62515 e41t47225 xlprg13-380ag xlprg40-31cgg xlpssrh03f03 xlpssrk13c10 xlpssrk14b06 xlpssrh01a07 xcdo385.2 xlpssrk09f06.1 xlpssrk02e02 e35t59112 e33t50133 e33t62460 xlpssrk12e03 xpsr901 e36t48595 e33t62113 e35t59575 e41t50240 xcdo1417 xlprg50-464ca xlpssrh08h05.3 xlprg24-460at xc600a e33t62620 xpsr540b e40t50334 xlpssrk08f05 xlpssrk12e06 xlpssrhxx285 xcdo36 e40t49173 xc472 xr738 xc556 xc847 http://www.biomedcentral.com/1471-2229/9/62 Figure rust resistance from other published studies Comparative mapping analysis between candidate R gene SNP loci mapped in the p150/112 population and QTLs for crown 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 F1(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 The location of the LpPc1 crown rust resistance locus is as described in [14] and marker nomenclature within the F1(SB2 × TC1) mapping population is described in [17] Page 17 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Perennial ryegrass NA 6-LG1 0.0 34.7 56.9 66.8 71.6 76.3 79.3 81.0 85.3 89.7 93.9 96.3 97.9 100.8 101.5 103.9 108.8 111.5 114.4 115.7 117.4 120.7 122.8 128.7 132.7 133.4 135.0 138.2 141.4 142.5 143.2 143.9 151.6 155.6 156.5 158.5 159.5 160.5 162.5 164.7 176.1 176.4 176.7 177.4 xlpap2b.2 xlprg1-369ct Perennial ryegrass Italian ryegrass Hexaploid oat xlpesi3f p150/112 - LG1 Barley - 1HS Rye - 1RS - LG1 LG 4_12 Wheat - 1AS xlpesi3e.1 xlplt16ab xlpssa.2 xlplt16aa 0.0 e33t50800 xHor2 5.0 0.0 xmwg938 cdo580-2(5) 0.0 xlpa05_071Qs292 avnA 0.0 5.7 xLrK10-A 4.4 xc1239Aa 8.9 xmwg206 6.0 xLrK10 LrK10 8.0 xlptrx-649ag xMla x(Glu-3)1A 5.1 XpLrk10-A 6.5 bcd1482b e41t47500 11.0 12.7 12.2 xlpbcd762-274at E32/M-CAGC185 xMla6 17.0 8.0 Xmwg837 LrK10 15.0 xcdo580 22.6 xlpcdo1173-212ac xmwg837 8.6 cdo1423a E32/M-ctag307 21.0 xlpssrk10f08 27.0 25.6 xHo21 10.2 xlpssrk10f08 cdo718b 29.0 P347(10) xlpssrhxx238 35.0 xlpsalta.2 cdo580 38.0 xlpssrk15h05 47.0 xpps0136c E35/M-CAAC47 43.0 28.5 xpps0094a xlpssrk03a02 cdo1173a 49.0 xpps0305a re4m2_7x 56.0 xcdo98 E32/M-CACT186 64.0 xlpcadc-561.45tg xlpssrk07f07 29.2 xlppsr168-699ag xlpssrk12d11 29.9 xlpcdo98-596ac E35/M-CTTC57 xlpssrh09e12.2 84.0 30.6 xlpf5h.2 pta71a 84.0 xlpssrk14d02 xlpssrk07f07-103at 31.3 cdo187 94.0 xlpssrh07g03 32.3 xlpcadc xlpssrk07f07 xlpssrh12g03 33.3 16-06G269 115.0 xlpssrk15h05 xpsr601 36.2 xpps0066b xbcd1072a xlpssrk08a06.2 xlpssrk10g04 41.8 xlpssrh12a04 xbcd738 xpsr162 xlppif 45.0 e41t47180 xlpssrh12g03 e33t50175 56.6 xlpthb-317ag xcdo202 89.7 xlposeindel xlpwalie e41t59188 93.5 xlpwalic-170ag xlpnox-1131at 122.6 xlp4cljb 152.1 e33t61133 xlpmtd xlprg8-271ct xlpssrk14c08 xlpwalic xlpssrk10g04 xpps0381a xpps0174a xpps0231a xpps0038b xlprg29-293ct xlpnox-1131at xlpnox-735ag xpps0211b Figure 10 Comparative mapping analysis of the perennial ryegrass LrK10 SNP locus (xlprg1-368ct) 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 Page 18 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 tion and implementation in forage grass improvement programs Authors' contributions PD carried out the experimental work and the majority of analysis, prepared the tables and figures and the primary drafts of the manuscript, and contributed to finalisation of the text and journal-specific formatting NC co-conceptualised the project and contributed to data analysis and text preparation TS provided EST sequence information and assisted preparation of files for GenBank submission TG co-conceptualised the project and contributed to text preparation KS co-conceptualised the project and developed and contributed genetically-defined plant materials GS provided EST sequence information and valuable editorial advice JF co-conceptualised the project, provided overall project leadership, and co-developed interim and final drafts of the manuscript Additional material Additional File http://www.biomedcentral.com/1471-2229/9/62 Additional File Functional characterisation of predicted translation products from perennial ryegrass candidate R genes All information was derived from Pfam links within the best-available Uniprot wuBLASTX hits in BASC Pfam information was used to obtain the probable location of candidate sequences, protein size, position of NBS sequence and number of LRR repeats Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S4.doc] Additional File Major protein sequence motifs in predicted Lolium NBS domains aMotifs listed in the order of occurrence in the NBS domain of putative perennial ryegrass R genes Perennial ryegrass motifs were named in accordance with descriptions obtained from both rice and A thaliana [30,31,66]; bBioinformatic analysis using Pfam on putative R gene sequences identified all to be CC-NBS types (CNL denotes CC-NBS-LRR and TNL denotes TIR-NBS-LRR); cConsensus amino acid sequences for Lolium NBS sequences were derived from MEME, while those for wheat were derived from [58] Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S5.doc] Degenerate oligonucleotide primers used for NBS domain-containing sequence amplification Sequence information for primer synthesis was obtained from published data specific to barley, sorghum and perennial ryegrass Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S1.doc] Additional File Additional File Additional File Bioinformatic (BLASTX and wuBLASTX) annotation of cloned and sequenced primary and secondary perennial ryegrass R gene templates to both GenBank and UniProt databases within the Bioinformatic Advanced Scientific Computing (BASC) database (as of June 2007 release) BASC is linked to the rice Ensemble Browser and Uniprot databases and employs known gene ontology and Pfam domain analysis to assign putative function to candidate sequences Nomenclature of paralogous sequences is based on the unique identifier for the primary template sequence (e.g., LpESTe11_14) followed by a numerical suffix (.1, etc.), e.g LpESTe11_14rg.1 Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S2.doc] Reference information for sequences corresponding to individual clusters identified during phylogenetic analysis for the complete NBS domain (P-Loop-GLPL), as depicted in Additional File Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S7.doc] Additional File Summary details for specific R gene-directed degenerate primer pair combinations, as described in Additional File 1, along with primer pair code, numbers of amplification products and corresponding R gene templates Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S3.doc] Summary information of amino acid structure for NBS domain protein sequence motifs numbered in Fig 2, based on matching using the MEME program Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S6.doc] Additional File NJ dendrograms based on amino acid alignment of the full-length (PLoop – GLPL) regions of NBS protein domains encoded by Lolium R genes Bootstrap values are displayed as percentages of 1000 neighbour joining bootstrap replications Bootstrap values at or greater than 65% are shown Bars at the right of the dendrograms represent R gene sub-classes Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S8.ppt] Additional File Reference information for sequences corresponding to individual clusters identified during phylogenetic analysis for the Kin-2A-GLPL region of the NBS domain, as depicted in Additional File 10 Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S9.doc] Page 19 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 http://www.biomedcentral.com/1471-2229/9/62 Additional File 10 NJ dendrograms based on amino acid alignment of the partial (Kin2A -GLPL) regions of NBS protein domains encoded by Lolium R genes Details are as described in the legend for Additional File Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S10.ppt] 10 Additional File 11 Summary information for LAP and SNuPe primers used for predicted R gene SNP validation Information on segregation structure, parental polymorphism, SNP variant and successful genetic map assignment is included All LAP PCRs and SNuPe reactions were designed for operating annealing temperatures of 55°C and 50°C, respectively Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S11.doc] Additional File 12 Comparative chromosomal positions of predicted putative orthologous R genes between perennial ryegrass and barley: Lps-217 (coded as xlprg50-464ca) on p150/112 LG2 compared to Hvs-217 at the bottom of chromosome 2H qLr represents a QTL for barley leaf rust resistance Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S12.ppt] 11 12 13 14 15 16 Additional File 13 Comparative chromosomal positions of predicted putative orthologous R genes between perennial ryegrass and barley: Lps-L8 (coded as xlprg54-688ag) on NA6-LG3 compared to Hvs-L8 at the bottom of chromosome 3H qMIL represents a major QTL for powdery mildew resistance in barley Click here for file [http://www.biomedcentral.com/content/supplementary/14712229-9-62-S13.ppt] 17 18 19 Acknowledgements 20 This work was supported by funding from the Victorian Department of Primary Industries, Dairy Australia Ltd., the Geoffrey Gardiner Dairy Foundation, Meat and Livestock Australia Ltd and the Molecular Plant Breeding Cooperative Research Centre (MPB CRC) 21 References 22 Fulkerson WJ, Slack K, Lowe KF: Variation in the response of Lolium genotypes to defoliation Aus J Agric Res 1994, 45:1309-1317 Terry RA, Tilley JMA: The digestibility of the leaves and stems of perennial ryegrass, cocksfoot, timothy, tall fescue, lucerne and sainfoin, as measured by an in vitro procedure J Brit Grassland Soc 1964, 19:363-372 Lancashire JA, Latch GCM: Some effects of crown rust (Puccinia coronata Corda) on the growth of two ryegrass varieties in New Zealand NZ J Agric Res 1966, 9:628-640 Price T: Ryegrass rust in Victoria Plant Protect Quart 1987, 2:189 Kimbeng CA: Genetic basis of crown rust resistance in perennial ryegrass, breeding strategies, and genetic variation among pathogen populations: a review Aus J Exp Agric 1999, 39:361-378 Mattner SW, Parbery DG: Rust-enhanced allelopathy of perennial ryegrass against white clover Agronomy J 2001, 93:54-59 23 24 25 26 Dracatos PM, Dumsday JL, Olle RS, Cogan NOI, Dobrowolski MP, Fujimori M, Roderick H, Stewart AV, Smith KF, Forster JW: Development and characterization of EST-SSR markers for the crown rust pathogen of ryegrass (Puccinia coronata f.sp lolii) Genome 2006, 49:572-583 Cagas B: The effect of infection by crown rust on the thousand-seed weight of annual ryegrass Rostlinna Vyroba 1986, 32:873-880 Landschoot PJ, Hoyland BF: Gray leaf spot of perennial ryegrass turf in Pennsylvania Plant Dis 1972, 76:1280-1282 Cornish MA, Hayward MD, Lawrence MJ: Self-incompatibility in ryegrass I genetic control in diploid Lolium perenne L Heredity 1992, 43:95-106 Clarke RG, Villata ON, Hepworth G: Evaluation of resistance to five isolates of Puccinia coronata f sp lolii in 19 perennial ryegrass cultivars Aus J Agric Res 1997, 48:191-198 Guthridge KM, Dupal MP, Kölliker R, Jones ES, Smith KF, Forster JW: AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.) Euphytica 2001, 122:191-201 van Treuren R, Bas N, Goossens PJ, Jansen J, van Soest LJM: Genetic diversity in perennial ryegrass and white clover among old Dutch grasslands as compared to cultivars and nature reserves Mol Ecol 2005, 14:39-52 Dumsday JL, Smith KF, Forster JW, Jones ES: SSR-based genetic linkage analysis of resistance to crown rust (Puccinia coronata f sp lolii) in perennial ryegrass (Lolium perenne) Plant Path 2003, 52:628-637 Armstead IP, Harper JA, Turner LB, Skøt L, King IP, Humphreys MO, Morgan WG, Thomas HM, Roderick HW: Introgression of crown rust (Puccinia coronata) resistance from meadow fescue (Festuca pratensis) into Italian ryegrass (Lolium multiflorum): genetic mapping and identification of associated molecular markers Plant Path 2006, 55:62-67 Muylle H, Baert J, Van Bockstaele E, Moerkerke B, Goetghebeur E, Roldan-Ruiz I: Identification of molecular markers linked with crown rust (Puccinia coronata f sp lolii) resistance in perennial ryegrass (Lolium perenne) using AFLP markers and a bulked segregant approach Euphytica 2005, 143:135-144 Muylle H, Baert J, Van Bockstaele E, Pertijs J, Roldan-Ruiz I: Four QTLs determine crown rust (Puccinia coronata f sp lolii) resistance in a perennial ryegrass (Lolium perenne) population Heredity 2005, 95:348-357 Studer B, Boller B, Bauer E, Posselt U, Widmer F, Kölliker R: Consistent detection of QTLs for crown rust resistance in Italian ryegrass (Lolium multiflorum Lam.) across environments and phenotyping methods Theor Appl Genet 2007, 115:9-17 Schejbel B, Jensen LB, Xing Y, Lübberstedt T: QTL analysis of crown rust resistance in perennial ryegrass under conditions of natural and artificial infection Plant Breed 2007, 126:347-352 Sim S, Diesburg K, Casler M, Jung G: Mapping and comparative analysis of QTL for crown rust resistance in an Italian × perennial ryegrass population Phytopathol 2007, 97:767-776 Dracatos PM, Cogan NOI, Dobrowolski MP, Sawbridge TI, Spangenberg GC, Smith KF, Forster JW: Discovery and genetic mapping of single nucleotide polymorphisms in candidate genes for pathogen defence response in perennial ryegrass (Lolium perenne L.) Theor Appl Genet 2008, 117:203-219 Curley J, Chakraborty N, Chang S, Jung G: QTL mapping of resistance to grey leaf spot in ryegrass: consistency of QTL between two mapping populations J Korean Turfgrass Sci 2008, 22:85-100 Studer B, Boller B, Herrmann D, Bauer E, Posselt UK, Widmer F, Kölliker R: Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.) Theoretical Appl Genet 2006, 113:661-671 Schejbel B, Jensen LB, Asp T, Xing Y, Lübberstedt T: Mapping QTL for resistance to powdery mildew and resistance gene analogues in perennial ryegrass Plant Breed 2008, 127:368-375 Curley J, Sim SC, Warnke S, Leong SB, Jung G: QTL mapping of resistance to grey leaf spot in ryegrass (Lolium perenne L.) Theor Appl Genet 2005, 111:1107-1117 Dracatos PM, Dumsday JL, Stewart A, Dobrowolski MP, Cogan NOI, Smith KF, Forster JW: Genetic diversity in Australasian populations of the crown rust pathogen of ryegrasses (Puccinia coronata f.sp lolii) In Molecular Breeding of Forage and Turf: The Page 20 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Proceedings of the 5th International Symposium on the Molecular Breeding of Forage and Turf Edited by: Yamada T, Spangenberg GC Springer, New York, USA; 2008:275-285 Aldaoud R, Anderson MW, Reed KFM, Smith KF: Evidence of pathotypes among Australian isolates of crown rust infecting perennial ryegrass Plant Breed 2004, 123:395-397 Dodds PN, Lawrence G, Pryor T, Ellis J: Genetic analysis and evolution of plant disease resistance genes In Molecular Plant Pathology Edited by: Dickinson M, Beynon J Sheffield Academic Press., Sheffield, UK; 2000:88-107 Dangl JL: Piece de Resistance: Novel class of plant disease resistance genes Cell 1995, 80:363-366 Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW: Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis Plant Cell 2003, 15:809-834 Zhou T, Wang Y, Chen JQ, Araki H, Jing Z, Jiang K, Shen J, Tian D: Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes Mol Genet Genom 2004, 271:402-414 Wisser RJ, Sun Q, Hulbert SH, Kresovich S, Nelson RJ: Identification and characterisation of regions of the rice genome associated with broad-spectrum, quantitative disease resistance Genetics 2005, 169:2277-2203 Madsen LH, Collins NC, Rakwalska M, Backes G, Sandal N, Krusell L, Jensen J, Waterman EH, Jahoor A, Ayliffe M, Pryor AJ, Langridge P, Schulze-Lefert P, Stougaard J: Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping Mol Genet Genomics 2003, 269(1):150-161 Cho J: Isolation and characterisation of resistance gene analogues (RGAs) in sorghum In Ph.D Thesis Texas A&M University, USA; 2005 Wenkai X, Mingliang X, Jiuren Z, Fengge W, Jiansheng L, Jingrui D: Genome-wide isolation of resistance gene analogs in maize (Zea mays L.) Theor Appl Genet 2006, 113:63-72 Monosi B, Wisser RJ, Pennill L, Hulbert SH: Full-genome analysis of resistance gene homologues in rice Theor Appl Genet 2004, 109:1434-1447 Irigoyen ML, Loarce Y, Fominaya A, Ferrer E: Isolation and mapping of resistance gene analogs from the Avena strigosa genome Theor Appl Genet 2004, 109:713-724 Li J, Xu Y, Fei S, Li L: Isolation, characterisation and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid Physiologia Plantarium 2006, 126:627-638 Ikeda S: Isolation of disease resistance gene analogues from Italian ryegrass (Lolium multiflorum Lam.) Grasslands Sci 2005, 51:63-70 Feuillet C, Keller B: High gene density is conserved at syntenic loci of small and large grass genomes Proc Natl Acad Sci USA 1999, 96:8265-8270 Cheng DW, Armstrong KC, Tinker N, Wight CP, He S, Lybaert A, Fedak G, Molnar SJ: Genetic and physical mapping of Lrk10-like receptor kinase sequences in hexaploid oat (Avena sativa L.) Genome 2003, 45:100-109 Forster JW, Cogan NOI, Dobrowolski MP, Francki MG, Spangenberg GC, Smith KF: Functionally-associated molecular genetic markers for temperate pasture plant improvement In Plant Genotyping II: SNP Technology Edited by: Henry RJ CABI Press, Wallingford, Oxford, UK; 2008:154-187 Feuillet C, Schachmayr G, Keller B: Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 resistance locus of wheat Plant J 1997, 11:45-52 Soreng RJ, Davis JI: Phylogenetics and character evolution in the grass family (Poaceae): Simultaneous analysis of morphological and chloroplast DNA restriction site character sets Botanical Rev 1998, 64:1-85 Jones ES, Mahoney NL, Hayward MD, Armstead I, Gilbert Jones J, Humphreys MO, King IP, Kishida T, Yamada T, Balfourier F, Charmet G, Forster JW: An enhanced molecular marker based genetic map of perennial ryegrass (Lolium perenne) reveals comparative relationships with other Poaceae genomes Genome 2002, 45:282-295 Sawbridge T, Ong E, Binnion C, Emmerling M, McInnes R, Meath K, Nguyen N, Nunan K, O'Neill M, O'Toole F, Rhodes C, Simmonds J, Tian P, Wearne K, Webster T, Winkworth A, Spangenberg G: Gen- http://www.biomedcentral.com/1471-2229/9/62 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 eration and analysis of expressed sequence tags in perennial ryegrass (Lolium perenne L.) Plant Sci 2003, 165:1089-1100 Erwin TA, Jewell EG, Love CG, Lim GAC, Li X, Chapman R, Batley J, Stajich JE, Mongin E, Stupka ER, Spangenberg G, Edwards D: BASC: an integrated bioinformatics system for Brassica research Nucleic Acids Res 2007, 35:870-873 Cogan NOI, Ponting RC, Vecchies AC, Drayton MC, George J, Dracatos PM, Dobrowolski MP, Sawbridge T, Smith KF, Spangenberg G, Forster JW: Gene-associated single nucleotide polymorphism discovery in perennial ryegrass (Lolium perenne L.) Mol Genet Genom 2006, 276:101-112 Leister D, Ballvora A, Salamini F, Gebhardt C: A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants Nat Genet 1996, 14:421-429 Faville MJ, Vecchies AC, Schreiber M, Drayton MC, Hughes LJ, Jones ES, Guthridge KM, Smith KF, Sawbridge T, Spangenberg GC, Bryan GT, Forster JW: Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.) Theor Appl Genet 2004, 110:12-32 Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer ELL: The Pfam protein families database Nucleic Acids Res 2002, 30:276-280 Finn RD, Mistry J, Scuster-Bockler B, Griffiths-Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Sonnhammer LL, Bateman A: Pfam: clans, web tools and services Nucleic Acids Res 2006, 34:D247-D251 Bailey TL, Elkan C: The value of prior knowledge in discovering motifs with MEME Proc Int Conf Intell Syst Mol Biol 1995, 3:21-29 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 1997, 24:4876-4882 Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap Evolution 1985, 39:783-791 Jones ES, Dupal MP, Dumsday JL, Hughes LJ, Forster JW: An SSRbased genetic linkage map for perennial ryegrass (Lolium perenne L.) Theor Appl Genet 2002, 105:577-584 Fulton TM, Chunwongse J, Tanksley SD: Microprep protocol for extraction of DNA from tomato and other herbaceous plants Plant Mol Biol Rep 1995, 13:207-209 Maleki L, Faris JD, Bowden RL, Gill BS, Fellers JP: Physical and genetic mapping of wheat kinase analogs and NBS-LRR resistance gene analogs Crop Sci 2003, 43:660-670 Gallego F, Feuillet C, Messmer M, Penger A, Graner A, Yano M, Sasaki T, Keller B: Comparative mapping of the two wheat leaf rust resistance loci Lr1 and Lr10 in rice and barley Genome 1998, 41:328-336 Dangl JL, Dietrich RA, Richberg MH: Death don't have no mercy: cell death programs in plant-microbe interactions Plant Cell 1996, 8(10):1793-1807 Dangl JL, Holub E: La Dolce Vita: a molecular feast in plantpathogen interactions Cell 1997, 91:17-24 Ellis J, Lawrence G, Ayliffe M, Anderson P, Collins N, Finnegan J, Frost D, Luck J, Pryor T: Advances in the molecular genetic analysis of the flax-flax rust interaction Ann Rev Phytopathol 1997, 35:271-291 Ellis J, Dodds P, Pryor T: Structure, function and evolution of plant disease resistance genes Curr Op Plant Biol 2000, 3:278-284 Dangl JL, Jones JD: Plant pathogens and integrated defence responses to infection Nature 2001, 411:826-833 Bai J, Pennill LA, Ning J, Lee SW, Ramalingam J, Webb CA, Zhao B, Sun Q, Nelson JC, Leach JE, Hulbert SH: Diversity in nucleotide binding site-leucine-rich repeat genes in cereals Genet Res 2002, 12:1871-1884 Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S, Sobral BW, Young ND: Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding site family Plant J 1999, 20:317-332 Meyers BC, Kaushik S, Nandety RS: Evolving disease resistance genes Curr Op Plant Biol 2005, 8:129-134 Leister D, Kurth DA, Laurie DA, Yano M, Sasaki T, Devos KM, Graner A, Schulze-Lefert P: Rapid reorganisation of resistance gene homologues in cereal genomes Proc Natl Acad Sci USA 1998, 95:370-375 Page 21 of 22 (page number not for citation purposes) BMC Plant Biology 2009, 9:62 69 70 71 72 73 74 75 76 http://www.biomedcentral.com/1471-2229/9/62 Xing Y, Frei U, Schejbel B, Asp T, Lübberstedt T: Nucleotide diversity and linkage disequilibrium in 11 expressed resistance genes in Lolium perenne BMC Plant Biol 2007, 7:43 Faris JD, Li WL, Liu DJ, Chen PD, Gill BS: Candidate gene analysis of quantitative disease resistance in wheat Theor Appl Genet 1999, 98:219-225 Li WL, Faris JD, Chittoor JM, Leach JE, Hulbert SH, Liu DJ, Chen PD, Gill BS: Genomic mapping of defense response genes in wheat Theor Appl Genet 1999, 98:226-233 Takahashi W, Fujimori M, Miura Y, Komatsu T, Nishizawa Y, Hibi T, Takamizo T: Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene Plant Cell Reps 2004, 23:811-818 Ponting RC, Drayton MC, Cogan NOI, Dobrowolski MP, Smith KF, Spangenberg GC, Forster JW: SNP discovery, validation, haplotype structure and linkage disequilibrium in full-length herbage nutritive quality genes of perennial ryegrass (Lolium perenne L.) Mol Genet Genom 2007, 278:585-597 Dracatos PM, Dobrowolski MP, Lamb J, Olle R, Gendall AR, Cogan NOI, Smith KF, Forster JW: Development of genetically homogenised populations of the crown rust pathogen (Puccinia coronata f.sp lolii) for disease trait dissection in perennial ryegrass (Lolium perenne L.) Australasian Plant Path 2009, 38:55-62 Dobrowolski MP, Forster JW: Linkage disequilibrium-based association mapping in forage species In Association Mapping in Plants Volume Chapter Edited by: Oraguzie NC, Rikkerink E, Gardiner SE, De Silva NH Springer, New York, USA; 2007:97-209 Miura Y, Hirata M, Fujimori M: Mapping of EST-derived CAPS markers in Italian ryegrass (Lolium multiflorum Lam.) Plant Breed 2007, 126:353-360 Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 22 of 22 (page number not for citation purposes) ... Smith KF, Forster JW: Discovery and genetic mapping of single nucleotide polymorphisms in candidate genes for pathogen defence response in perennial ryegrass (Lolium perenne L.) Theor Appl Genet... disease resistance QTLs SNP mapping of two candidate R genes in both the F1(NA6 × AU6) and p150/112 mapping populations has provided possible candidates for the major gene crown rust resistance. .. diversity in motif content within the NBS domain and grouping of specific motifs into sub-classes based on shared sequence origin Phylogenetic analysis of perennial ryegrass R genes Phylogenetic