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BMC Plant Biology
Open Access
Research article
Molecular characterisation and genetic mapping of candidate genes
for qualitative disease resistance in perennial ryegrass (Lolium
perenne L.)
Peter M Dracatos
1,2,4
, Noel OI Cogan
1,4
, Timothy I Sawbridge
1,4
,
Anthony R Gendall
2
, Kevin F Smith
3,4
, German C Spangenberg
1,4
and
John W Forster*
1,4
Address:
1
Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, 1 Park Drive, La Trobe University
Research and Development Park, Bundoora, Victoria 3083, Australia,
2
Department of Botany, Faculty of Science, Technology and Engineering, La

Trobe University, Bundoora, Victoria 3086, Australia,
3
Department of Primary Industries, Biosciences Research Division, Hamilton Centre, Mount
Napier Road, Hamilton, Victoria 3300, Australia and
4
Molecular Plant Breeding Cooperative Research Centre, Bundoora, Victoria, Australia
Email: Peter M Dracatos - ; Noel OI Cogan - ;
Timothy I Sawbridge - ; Anthony R Gendall - ;
; German C Spangenberg - ;
John W Forster* -
* Corresponding author
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-for-

gene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance.
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: />© 2009 Dracatos et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Plant Biology 2009, 9:62 />Page 2 of 22
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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, digestibil-
ity, palatability and the ability to recover from heavy defo-
liation 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 (Mag-
naporthe grisea), dollar spot (Sclerotinia homoeocarpa) and
brown patch (Rhizoctonia solani) reduce turf quality [9].
The development of cultivars resistant to each of these dis-
eases 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 quantita-
tive trait loci (QTLs) for disease resistance have been
detected in Lolium species for resistance to crown rust [14-
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 popu-
lations [26] is consistent with the presence of different vir-
ulence 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 N-
terminus and a leucine-rich repeat (LRR) domain
involved in the host recognition of pathogen-derived elic-
itors. 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 dis-
tributed non-randomly throughout the genome [30-32].
Clustering of R genes is known to facilitate tandem dupli-
cation of paralogous sequences and generation of new
resistance specificities to counter novel avirulence deter-

minants in evolving pathogen populations [30-34].
NBS domain-containing sequences have been isolated
using degenerate PCR from many agronomically-impor-
tant Poaceae species including cereals [33-37] and forage
grasses [24,38,39]. In a comparison with the fully-
sequenced 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 pro-
vide 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 cul-
tivars, and putative Lrk10 ortholoci are structurally con-
served 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 confer-
ring 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 Ave-
neae 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 func-
tional annotation, motif structure classification and phyl-
ogenetic analysis. Single nucleotide polymorphisms
(SNPs) were discovered through re-sequencing of haplo-
types 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)
BMC Plant Biology 2009, 9:62 />Page 3 of 22
(page number not for citation purposes)
system [47]. Each EST was functionally annotated using
data from microarray-based transcriptomics experiments,
the rice Ensembl browser, Pfam and gene ontology data-
bases. 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 spe-
cies (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 previ-
ously 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 resist-
ance 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 orien-
tation) 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 inter-
pretation 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 analy-
sis against the GenBank and the Uniprot databases,
respectively. Genomic DNA sequences were translated to
amino acid sequences using Transeq software. Each pep-
tide 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 Elic-
itation (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 rea-
ligned 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 par-
ents and progeny of the F
1
(NA
6
× AU

6
), Vedette
6
and
p150/112 [45,56] mapping families using the CTAB
method [57]. A genotypic panel for genetic map assign-
ment was constructed of 141 F
1
(NA
6
× AU
6
) and 24 p150/
112 F
1
genotypes as previously described [21].
In vitro discovery, validation and mapping of gene-
associated 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 nucle-

otide 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 heterol-
ogous 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]. Chro-
mosomal locations of LrK10 ortholoci were compared
between Lolium and Avena species using common heterol-
BMC Plant Biology 2009, 9:62 />Page 4 of 22
(page number not for citation purposes)
ogous RFLP loci [58]. Further comparative genomic anal-
ysis 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 heterolo-
gous PCR and degenerate PCR, and a bioinformatic dis-
covery 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 popu-

lation 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 (cor-
responding to 99 NBS-containing genes and 3 receptor
kinase genes) were annotated (Additional File 2) and sub-
jected 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 candi-
date gene discovery process and further applications is
depicted in Figure 1.
In the empirical approach category, translational genom-
ics 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 tem-
plate 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 genesFigure 1
Schematic representation of empirical and bioinformatics-based discovery of perennial ryegrass R genes. Sub-
sequent bioinformatic analysis leads to two streams of genetic analysis, including sequence characterisation, in vitro SNP discov-
ery and large-scale genetic mapping.

Cereal R gene
ortholoci
Degenerate primers
oligonucleotide
Perennial ryegrass
ESTs and
GenBank-derived clones
Bioinformatic
annotation for
functional role
Amino acid
alignment
R-gene characterisation
and classification
into distinct
classes and families
Candidate resistance
gene discovery
Motif analysis
SNP discovery
in F
1
(NA
6
xAU
6
)
parental genotypes
SNP validation
and

genetic mapping
Association of SNPs with
resistance loci in
perennial ryegrass
and cereal species
R gene sequence similarity and
relationship to macrosynteny
BMC Plant Biology 2009, 9:62 />Page 5 of 22
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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
Primer design based on Pooideae R gene templates
LpLrk10 Wheat leaf rust receptor kinase [41]
LpPcaClone1 Oat NBS-LRR candidate from Pca cluster [37]
LpPcaClone2 Oat NBS-LRR candidate from Pca cluster
LpPcaClone3 Oat NBS-LRR candidate from Pca cluster
LpPcaClone4 Oat NBS-LRR candidate from Pca cluster
LpHvClone1 Barley NBS-LRR co-locating with QTL [33]
LpHvClone2 Barley NBS-LRR co-locating with QTL
LpHvClone3 Barley NBS-LRR co-locating with QTL
LpHvClone4 Barley NBS-LRR co-locating with QTL
LpHvClone5 Barley NBS-LRR co-locating with QTL
Degenerate primer pair design
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 primer pair
LpNBS-LRR1 Degenerate primer pair
LpNBS-LRR2 Degenerate primer pair
LpNBS-LRR3 Degenerate primer pair
LpNBS-LRR4 Degenerate primer pair
LpNBS-LRR5 Degenerate primer pair
LpNBS-LRR6 Degenerate primer pair
LpNBS-LRR7 Degenerate primer pair
LpNBS-LRR8 Degenerate primer pair
LpNBS-LRR9 Degenerate primer pair
LpNBSC1 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
Primer design for candidate Lolium R genes
LpESTa03_10rg LpEST from bioinformatic discovery [46]
LpESTa08_14rg 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
BMC Plant Biology 2009, 9:62 />Page 6 of 22
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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 fre-
quency of inosines (>15%) and predominantly amplified
retrotransposon-like sequences (data not shown). In con-
trast, combinations of largely non-degenerate primer
pairs based on sequence information from multiple
Poaceae species (barley, sorghum and ryegrass) (Addi-
tional File 1), successfully generated NBS domain-con-
taining 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 < 1 × 10
-15

) to known R genes (Additional
File 2).
Database searches for previously-characterised ryegrass
NBS sequences identified 51 accessions from Italian rye-
grass-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 gener-
ated single amplicons of the expected size (Table 1, Addi-
tional 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 dif-
ferent 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 con-
taining sequences were observed. Of the different sub-

classes 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 com-
pared 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 con-
served within the Poaceae, and the Kin-2A and RNBS-II
motifs were the most conserved among the CC-NBS
LpESTg01_20rg LpEST from bioinformatic discovery
LpESTg04_17rg LpEST from bioinformatic discovery
LpESTg06_13rg LpEST from bioinformatic discovery
LpESTh04_17rg LpEST from bioinformatic discovery
LpESTh05_28rg LpEST from bioinformatic discovery
LpESTh07_17rg LpEST from bioinformatic discovery
LPCL_38150 LpEST from bioinformatic discovery
LPCL_8913 LpEST from bioinformatic discovery
LpHvESTClone1 LpEST from bioinformatic discovery
LpHvESTClone2 LpEST from bioinformatic discovery
LpHvESTClone3 LpEST from bioinformatic discovery
LpHvESTClone4 LpEST from bioinformatic discovery
LpAG205017 RG sequence from Italian ryegrass [39]
LpAG205018 RG sequence from Italian ryegrass
LpAG205035 RG sequence from Italian ryegrass
LpAG205050 RG sequence from Italian ryegrass

LpAG205055 RG sequence from Italian ryegrass
LpAG205063 RG sequence from Italian ryegrass
LpEST = perennial ryegrass EST; RG = resistance gene.
Table 1: Classification of primary R gene templates used for host-specific genetic analysis, according to isolation strategy (Continued)
BMC Plant Biology 2009, 9:62 />Page 7 of 22
(page number not for citation purposes)
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 sub-
classes 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-2A-
GLPL). Unrelated NBS domain sequences from A. thal-
iana, 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 ana-
lysed. 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 puta-
tive orthologues identified from preliminary BLASTX
analysis. The majority were most closely related to those
from other ryegrass species, although some showed high-
est sequence similarity to template genes from other spe-
cies. 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 spe-
cies, which were not included within the alignment.
Eight major clusters were identified for the Kin-2A-GLPL
region (Additional File 9, Additional File 10). Ryegrass-
derived 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 dicotyledo-
nous plant species were separately clustered for the P-
Loop-GLPL [cluster E], but co-located in several distinct
clusters [cluster D and E] with ryegrass-derived sequences
for the Kin-2A-GLPL region.
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 ana-
lysed and a total of 819 R gene SNPs were predicted, at an
overall frequency of 1 per 46 bp. A total of 11 (17%) tem-
plate 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 sam-
ple set. SNP frequencies within parental genotypes was
higher for NA
6
(38) than for AU
6
(20). A further 8 SNPs
with biparental (AB × AB) segregation structures and 4
SNPs with AA × BB structures were identified.
Multiple R gene SNPs from 37 (69%) of 54 SNP-contain-
ing R gene contigs were validated (Additional File 11). A
total of 26 R genes were assigned to loci on the parental
maps of the F
1
(NA
6
× AU

6
) mapping population (22 on
all NA
6
LGs [Figs. 3, 4], 10 on all but LG4 for AU
6
[Figs. 5,
6]). SNPs in four R gene loci showed biparental segrega-
tion 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 F
1
(NA
6
× AU
6
) (Fig.
7)
R gene locus clusters were identified on a number of LGs,
often in close proximity to mapped DR gene loci (repre-
sented 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. Coinci-
dences 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 fur-
ther two loci were assigned to the centromeric region of
p150/112 LG2, 4 cM distant from the genomic DNA-
derived 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 com-
parative alignment, with the hexaploid oat Pca cluster on
BMC Plant Biology 2009, 9:62 />Page 8 of 22
(page number not for citation purposes)
Representation of motif patterns in the NBS domain of perennial ryegrass R gene sequencesFigure 2
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).
NBS ID BLASTP NBS motif structure
a
LpHvESTClone4.3 3e
-170


21

43

16


8

22


49


17


10


14

2

4 39

15

31



LpESTe11-14rg.2 4.6e
-161


21

43

16


8

22


49

17


10


14


2

4 39

15

31



AsPcaClone1 2.5e
-136



30


9 5

18


11

6

47


7

29

1

28


9 5

18


11

6

47

7

29


LpPcaClone4.1 3.5e
-129


30


9 5

18


11

6

47


7

29

1

28



LpPcaclone1 1.2e
-104


30


9 5

18


11

6

47


7

29

LpRG2NBS 3e
-102


27

42

40

12

15

22

35


Lpd02_gp08.1 2.8e
-100



3 42

40

12

36

33

35


LpESTa10_13rg.2 2.5e
-92



20


24

33

35

25


48


29 45

28

LpESTe11-14rg.3 2.9e
-81



37

26

13

8 19

20

36


LpRGcontig1 1.6e
-76


27


23

12


36


33


34

17



LpPcaClone3.1 3.9e
-75



18


11


25

7

29

1

28



LpNBS-LRR4 4e
-74



50


26

44


34


31


32

16


41


LpNBSC2 4.2e
-72


27

23

20

24

46

38


Lpa11_gp09 7.8e
-69

27

23


12


36


33


34

17



LpESTa03_10rg.2 6.9e
-67



37


41 26

13


3

19


LpNBSC8 4.1e
-66


27


23

20


24


46


38

17



LpRGcontig3 2.7e
-54


27


19


12


36


49


33


17


LpNBSC10 1.5e

-51



13

23

20

24

33

17


LpHvESTClone1.1 9.2e
-43



3

19

32

12

LpNBSC6 3.9e
-39



20


24


46


38


17


29 45

28



LpESTh05_28rg.1 1.3e
-32

25


48


29

45

31


49



Lpd07_gp09 1.4e
-20



27

23

18

12

36

17



60
90
120
150
180
210
240
270 300
30
0
Amino acid residues
BMC Plant Biology 2009, 9:62 />Page 9 of 22
(page number not for citation purposes)
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
F
1
(NA
6
× AU
6
)
progeny
LG location and
mapped locus
coordinate (cM)
[F
1
(NA
6
× AU
6
)]
LG location and
mapped locus
coordinate (cM)
[p150/112 population]
LpLrK10.1 rg1 15/1500 107 1 NA
6

-LG1- 34.7 N/A
LpPcaClone 1.1 rg2 3/358 203 0 N/A N/A
LpPcaClone 1.2 rg3 0/470 N/A 0 N/A N/A
LpPCAClone2.1 rg4 0/380 N/A 0 N/A N/A
LpPcaClone3.1 rg5 1/510 510 0 N/A N/A
LpPcaClone3.2 rg6 0/187 N/A 0 N/A N/A
LpPcaClone3.3 rg7 0/187 N/A 0 N/A N/A
LpPcaClone4.1 rg8 1/510 510 1 NA
6
– LG1- 151.6 N/A
LpHvclone1 rg9 0/250 N/A 0 N/A N/A
LpHvclone2 rg10 0/230 N/A 0 N/A N/A
LpHvclone3 rg11 2/406 N/A 0 N/A N/A
LpRGContig1 rg12 4/646 162 1 N/A N/A
LpRGContig2 rg13 2/504 86 1 AU
6
– LG2- 57.4 LG2 – 32.5
LpRGContig3 rg14 0/590 N/A 0 N/A N/A
LpRG1NBS rg15 18/410 30 3 NA
6
– LG2- 172.7 N/A
LpRG2NBS rg16 17/412 24 2 N/A N/A
LpRG3NBS rg17 14/540 36 0 N/A N/A
LpRG4NBS rg18 4/537 134 0 N/A N/A
LpRG5NBS rg19 20/520 26 0 N/A N/A
LpRG6NBS rg20 25/540 22 0 N/A N/A
LpRG7NBS rg21 15/520 35 0 N/A N/A
LpNBSC5 rg22 3/423 141 1 N/A N/A
LpESTa03_10rg.1 rg23 23/295 77 3 AU
6

– LG1- 74.1 N/A
LpESTa08_14rg rg24 24/723 15 2 NA
6
– LG2- 166.6 LG2 – 62.9
LpESTa10_13rg rg25 5/594 119 1 N/A N/A
LpESTb06_11rg rg26 10/729 45 3 AU
6
– LG5 – 65.1 LG5 – 20.2
LpESTc10_19rg rg27 9/550 96 2 NA
6
– LG5 – 0.0;
AU
6
– LG5 – 68.4
LG5 – 19.2/19.7
LpESTd08_13rg rg28 6/859 61 2 NA
6
– LG5 – 10.9;
AU
6
– LG5 – 27.0
N/A
LpESTe11_14rg.1 rg29 14/684 49 1 NA
6
– LG1 – 176.1 N/A
LpESTe11_14rg.2 rg30 14/684 49 2 AU
6
– LG1 – 118.9 N/A
LpESTe11_14rg.3 rg31 12/591 66 2 NA
6

– LG2 – 161.5 N/A
LpESTe11_14rg.4 rg32 16/605 38 1 N/A N/A
LpESTe11_14rg.5 rg33 1/645 645 0 N/A N/A
LpESTe11_14rg.7 rg34 0/375 N/A 0 N/A N/A
LpESTe11_14rg.8 rg35 0/435 N/A 0 N/A N/A
LpESTe11_14rg.9 rg36 3/423 141 0 N/A N/A
LpESTe11_14rg.10 rg37 8/380 48 0 N/A N/A
LpESTe11_14rg.11 rg38 0/690 N/A 0 N/A N/A
LpESTe11_14rg 12 rg39 7/810 116 0 N/A N/A
LpESTf06_19rg.1 rg40 9/550 61 3 NA
6
– LG2 – 71.8/
78.0
LG2 – 32.5
LpESTf11_11rg rg41 35/890 25 0 N/A N/A
LpESTg01_20rg rg42 3/325 252 2 AU
6
– LG3 – 45.9 N/A
LpESTg04_17rg.1 rg43 7/670 26 3 NA
6
– LG4 – 92.8/
94.9
N/A
LpESTg06_13rg rg44 59/880 49 1 NA
6
– LG2 – 164.4 N/A
LpESTg10_13rg.1 rg45 14/540 143 2 NA
6
– LG5 – 37.1 N/A
LpESTg10_13rg.2 rg46 2/540 270 2 NA6 – LG7 – 0 N/A

LpESTh04_17rg rg47 12/604 8 3 AU
6
– LG6 – 137.7 N/A
LpESTh05_28rg.1 rg48 13/580 108 2 AU
6
– LG5 -0.0 N/A
LPCL_8913 rg49 8/600 39 3 NA
6
– LG6 – 134/
134
N/A
LpHvESTClone1.1 rg50 90/730 73 3 N/A LG2 – 55.2
LpHvESTClone1.2 rg51 2/550 225 0 NA
6
– LG5 –
105.1/124.8
N/A
BMC Plant Biology 2009, 9:62 />Page 10 of 22
(page number not for citation purposes)
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 interspe-
cific hybrid ψ-F
2
(MFA × MFB) population map, but a lim-
ited number of common markers precluded further

interpretation (data not shown).
Comparative genomic analysis detected conserved rela-
tionships 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).
Discussion
Large-scale survey of perennial ryegrass NBS domain-
containing 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 degen-
erate primer-based amplification. In this study, 102 dis-
tinct 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 dif-
ferent selection pressures of surrounding pathogen popu-
lations. 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 spe-
cific to non-TIR sequences were able to amplify PCR prod-
ucts 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 con-
tained 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. thal-
iana [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
LpHvESTClone1.3 rg52 6/556 93 0 N/A N/A

LpHvESTClone1.4 rg53 60/690 12 0 N/A N/A
LpHvESTClone2.1 rg54 26/670 66 3 NA
6
– LG3 – 130.8 N/A
LpHvESTClone3.1 rg55 98/1100 11 1 N/A N/A
LpHvESTClone4.1 rg56 3/646 215 1 N/A N/A
LpHvESTClone4.2 rg57 13/680 52 0 N/A N/A
LpHvESTClone4.3 rg58 0/270 N/A 0 N/A N/A
LpHvESTClone4.4 rg59 8/930 116 0 N/A N/A
LpAG205017 rg60 7/540 75 2 NA
6
– LG7 – 46.5;,
AU
6
– LG7 – 45.9
N/A
LpAG205018 rg61 7/601 13 2 AU
6
– LG1 – 187.4 N/A
LpAG205035 rg62 10/660 23 2 NA
6
– LG3 – 37.8 N/A
LpAG205050 rg63 14/610 44 1 N/A N/A
LpAG205055 rg64 10/664 86 3 NA
6
– LG2 –
149.4; AU
6
– LG2
– 86.9

N/A
LpAG205063 rg65 7/602 86 1 NA
6
– LG2 – 134.3 N/A
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.
Table 2: Summary information for in vitro SNP discovery and genetic mapping of candidate R gene SNPs (Continued)
BMC Plant Biology 2009, 9:62 />Page 11 of 22
(page number not for citation purposes)
were missing or duplicated. This suggests that the RNBS-I
and RNBS-II motifs may either play a role in pathogen-
specific recognition, or be less functionally significant
than other, more highly conserved domains mediating
resistance in plant species [30,66]. Alternatively, the pres-
ence 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 demon-
strate that TIR-NBS sequences from flax and A. thaliana
group in a separate cluster, as observed in a previous phy-
logenetic 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
) crossFigure 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 per-
enne 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 des-
ignated according to the nomenclature xlp-gene name abbreviation-nucleotide coordinate-SNP identity [48]. For instance, xlp-
chijb-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 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.
xlpap2b.2
0.0
xlprg1-369ct
34.7
xlpesi3f
56.9
xlpesi3e.1 xlplt16ab66.8
xlpssa.2 xlplt16aa
71.6
xlpa05_06Ws292
76.3

xlptrx-649ag xlpssrk12d11-038cg
79.3
xlpbcd762-274at
81.0
xlpcdo1173-212ac xlpzta-299ct
xlpssrk10f08
85.3
xlpsalta.289.7
xpps0136c
93.9
xpps0094a
96.3
xpps0305a
97.9
xlpcadc-561.45tg
100.8
xlppsr168-699ag101.5
xlpcdo98-596ac
103.9
xlpf5h.2
108.8
xlpssrk07f07-103at
111.5
xlpcadc
114.4
xlpssrk07f07
115.7
xlpssrk15h05
117.4
xpps0066b120.7

xlpssrk08a06.2
122.8
xlpssrh12a04 xlpssrh11g05
128.7
xlpp if
132.7
xlpssrh12g03
133.4
xlpthbna-317ag
135.0
xlposeindel
138.2
xlpwalie141.4
xlpwalic-170ag xlpchihs.1
142.5
xlp4cljb
143.2
xlpmtd
143.9
xlprg8-271ct
151.6
xlpssrk14c08155.6
xlpwalic
156.5
xlpssrk10g04
158.5
xpps0381a
159.5
xpps0174a xpps0055a
160.5

xpps0231a
162.5
xpps0038b
164.7
xlprg29-293ct176.1
xlpnox-1131at
176.4
xlpnox-735ag
176.7
xpps0211b xpps0114a
177.4
NA
6
-LG1
xpps0154a0.0
xpps0259c
8.8
xlppera-1041ag
16.4
xlpper1
21.1
xlplt16ba
36.0
xlpwalib
41.4
xlpwalih
42.5
xlppkabab49.5
xpps0122c
66.1

xlprg40-31cgg
71.8
xlprg40-284ag
78.0
xpps0410b
85.1
xpps0223b85.8
xpps0113b
86.5
xlpssrk14b01.2
90.2
xlpssrk09g05.2
91.6
xlpssrhxx050
93.5
xlpssrk03b03.2
94.9
xlpssrk05h02.2
97.4
xlpssrk09f06.199.3
xlphish3-282cg
106.2
xlphish3
112.2
xpps0153a
117.2
xpps0037c
118.7
xpps0328a
128.1

xlpssrk12e03
131.3
xlprg65-202gt134.3
xlpera
139.8
xlpera-376ct
143.2
xpps0080a
145.2
xlprg64-81at
149.4
xpps0400a151.5
xpps0439a
157.8
xlprg31-490ct
161.5
xlprg44-514ct
164.4
xlprg24-345ct
166.6
xlprg15-277gt
172.7
xlptc101821-122ct
185.6
xlptc116908-050ct189.3
xlptc89057-116ct
191.0
xlptc32601-503ac
196.4
NA

6
-LG2
xlpb07_06Ws249
0.0
xlpph
2.5
xlpssrk03g0510.6
xlpmtn
21.6
xlpmtc.1 xlpmtl.2
24.8
xlpmtj.1
28.6
xpps0007b xlpssrhxx242
31.4
xlpssrk08b01.136.2
xlprg62-159ag
37.8
xlpzta
40.3
xpps0177c xpps0373a
41.0
xpps0051a xlpcadd
41.7
xlpssrk09f08
42.6
xlpssrk09g05.1
44.4
xlpssrk12h01.347.1
xlpssrh02d12

51.1
xpps0039b
58.5
xpps0145b
66.8
xlpc4h.1
68.4
xlpcysme
73.0
xlppl b
78.4
xpps0353b84.1
xpps0213b xpps0164a
96.4
xpps0375a
98.5
xlpmads1
104.7
xlphak1
106.3
xlphak1-160cg118.4
xlpnvg
121.5
xlpcwnv xlpf5h.1
123.5
xlpnvc
125.5
xpps0322b
129.9
xlpssa.1 xlprg54-688ag

130.8
NA
6
-LG3
xpps0006d
0.0
xlpssrh03a08.2
3.3
xlpcell xpps0150a6.5
xlpcluster404
15.8
xlpssrk15f05.2
30.3
xpps0146b
34.0
xpps0423a
40.5
xpps0201b44.7
xpps0205b
47.9
xlpssrk05a11.1
50.4
xlpssrk08b11.1 xlpasra2
52.3
xlpasra2-132ag
53.7
xlpa22-201ct
54.4
xlpssrk01g06 xlphaka
55.1

xpps0018a xlpssrk03c05
xlpc hi e
56.4
xpps0439d
59.6
xpps0433b
62.8
xpps0202a
68.0
xlpzba
70.6
xlpssc xlpa22c
72.7
xlpkabaa-858.34ct75.5
xlp4clja
78.3
xlp4clja-323ag
81.9
xlpomt3.1
84.9
xlprg43-403ct
92.8
xlprg43-271ct94.9
xlpssrk07c11
98.8
xlpffta.1
106.3
NA
6
-LG4

xlpoxo-123cg
xlpcat-561cg
BMC Plant Biology 2009, 9:62 />Page 12 of 22
(page number not for citation purposes)
that this region may be more conserved across taxa. Con-
sensus 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. thal-
iana 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 com-
pared to 5 separate clusters identified in a previous study
[38]. Analysis of the larger number of Kin-2A-GLPL inter-
val 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 sub-
families. Sequences amplified from oat templates clus-
tered together with ryegrass template-derived R genes, sug-
gestive 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 mar-
ginally lower than that detected within a sub-set of 11 per-
ennial 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, possi-
bly due to paralogous sequence alignment. Large numbers
of haplotypes have been reported for other perennial rye-
grass 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
) crossFigure 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.
xlpsalta.1
0.0
xpps0463b
5.3
xlpssrh02h05
13.8
xpps0013a
20.8
xpps0432b
22.1
xpps0210b xpps0098d22.7
xlpomt2
25.8
xlphbd.2 xlpspsf
xlpcta-186ct xlp4cl3-1643.201ct
28.1
xlpssrk05h01 xlpssrk01c04
29.3
xlpdhna.232.0
xlpdhna.1
33.3
xlpctaa
35.8
xlpssrk11g12
40.4

xlphba.1
40.6
xlpdefa
44.4
xpps0374c
50.5
xpps0031b53.2
xlpdefa-233ct
57.1
xpps0022a
66.3
xpps0450a
69.5
xlpssrk12h01.5
77.3
xlpssrk11c07
80.2
xpps0019b
84.2
xlpssrk10b0786.8
xlpssrh09e12.1
87.5
xlpcha
91.6
xlpunk1-278ca
94.5
xlpccra
99.8
xlpdfrb103.8
xlpssrh02e01

108.8
xlpglr-1435ct
111.7
xpps0299c
114.2
xlprg49-105ca
xlprg49-275ct
134.0
NA
6
-LG6
xlpccoaomt1
0.0
xlpccoaomt1-100tg1.1
xlphba.2
2.2
xlpca
16.6
xlpcana-299ca
19.4
xlpsucsyn
22.1
xpps0473a
26.2
xlpcadlke04-98ct
28.9
xlpcadlke11-86tg31.6
xlpleaa-164ct
34.3
xlpssrk14b01.1

37.0
xpps0282b
39.4
xpps0049b
43.1
xpps0425b44.1
xpps0441a xpps0376c
46.1
xlprg60-216gt
46.5
xpps0131b
46.9
xpps0065b
47.7
xpps0466c
50.0
xpps0294b
53.4
xlpccha-284ga55.1
xlpznfcon3-489ct
58.8
xpps0429a xpps0447b
60.2
xlpssrh03a08.3
68.8
xlpssrh08h05.2
72.0
xlpa22a.1
75.8
xlpa22a.2

81.0
xlpthc.187.9
xlpthc.2
91.1
xlpmads4.2
104.3
NA
6
-LG7
NA
6
-LG5
xlprg27-743ct
xlprg27-912ag
0.0
xlprg28-509ct
10.9
xlprg45-196ct37.1
xlpcena xlpchijb-240cg
38.5
xlpdefc
39.2
xlpdeff
42.7
xlpes3g
63.9
xlpes3h xlphbb-210.230tg65.7
xlpssrk03b03.1
67.0
xlpssrk11g10

69.9
xlpssrk14c12
72.2
xlptla
81.3
xpps0032b
105.1
xpps0056a
124.8
xpps0111b125.6
xpps0149a
141.8
xpps0199d
144.5
xpps0397c
145.9
xlprg46-86gt
xlprg51-464ac
BMC Plant Biology 2009, 9:62 />Page 13 of 22
(page number not for citation purposes)
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 sub-
species 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 associa-
tion with DR gene SNP and RFLP loci [21,50]. QTL based
analysis and genetic mapping in wheat identified co-loca-
tion 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 signifi-

cance, 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 per-
ennial ryegrass trait-specific mapping populations [17-19]
but the limited number of common markers limits accu-
rate extrapolation between genetic maps. Two QTLs of
large magnitude were identified in each of the three pop-
ulations, 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
) crossFigure 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.
103.1
105.9
108.7
110.5
114.3
118.9
144.0
153.2
187.4
xlpap2b.1
0.0
xlpap2b.2
4.9
xlpap2-305ta
37.2
xpps0136a63.1
xlpesi3
70.4
xlpssrk10f08
71.8

xlprg23-177ag xlprg23-337ct
74.1
xlpssrk03a02-156ct
74.5
xlplt16aa74.9
xlpssrk15h05-027ct
75.7
xlpssrk15h05
76.5
xlpssrk08b01.2
77.8
xlpsalta.1
78.4
xlplt16ab
78.6
xlpesi3e
78.8
xlpssrk08b11.279.0
xlp4cljb xlppsr168-388at
79.1
xlpssrk12d11-038cg
79.7
xpps0401a
80.3
xpps0094b
82.0
xlptrx-540ct
84.0
xpps0255y
88.1

xlposeb-896ct xlpssrh09e12.3
xlpssrk07f07
90.2
xlposeaindel
91.0
xlpmtd
95.1
xlppif.2
99.7
xlpssrh12g03 xlpssrh12a04
xlpthb
xlpssrk12h01.2
xlpssrh09e12.2
xpps0038a
xpps0114b xpps0286a
xlprg30-707ag
xpps0411a
xlppcsa
xlprg61-23ag
xpps0037b
49.3
xpps0410a50.1
xlpssrk02e02
52.2
xlppkabab
55.0
xlprg13-380ag
57.4
xlpssrk02d08.2
xlpssrk09f06.1

61.8
xlphst3
74.1
xlpwesr5a
83.5
xlprg64-81at
xlpera
86.9
xpps0080b
91.0
xlptc32601-514gt
99.8
xlptc101821-ct
103.6
xlptc116908-061ag
107.2
xlptc89057-072ct108.6
xlphcd266-096cg
111.1
xpps0172b
113.6
xpps0347a
120.4
xpps0121b
0.0
xpps0259b
15.6
xlpssrk02c09.220.9
xlpssrh05f02
21.7

xlpap2b.3
23.5
xpps0218a
26.8
xlpper1.2
30.4
xpps0220b35.2
xpps0122a
44.5
xpps0153b
45.9
xpps0223c
46.6
xpps0333a
48.0
xpps0113c
48.7
xlpsfta-404ct
0.0
xlpsfta.1 xlpsfta.2
1.2
xlpssrk09f06.96
6.9
xpps0198a
9.9
xlpc4h.2
14.0
xlpmtn xlpmtg
24.1
xlprg42-331ag

45.9
xlpssrh06h02
67.8
xpps0439b
72.5
xlpssrh03h02 xlpssrh02f02
73.2
xlpmtc xlpmtl.1
81.2
xlpmtl.2
82.7
xlpssrk14b01.3
88.3
xlpmtc-114.11ag91.3
xlpssrhxx242
93.5
xpps0007a xpps0373b
94.2
xlpssrk09f08
94.8
xlpssrh02c11
95.4
xlpssrk14f1296.4
xlpssrh02d12
97.4
xlpssrk15f05.1
101.4
xlpwrky14-166ga
104.2
xpps0164c

109.1
xlphak1f80r
120.3
xlphak1
123.0
xlpssrk07h08126.2
xlpcwinv1 xlpinvg
151.7
AU
6
-LG1
AU
6
-LG2
AU
6
-LG3
AU
6
-LG4
xpps0276a
0.0
xpps0006a
9.1
xlpcell
22.4
xlpoxo-123ga
25.8
xpps0423c
46.3

xpps0128b
47.9
xpps0205c xpps0201c
49.2
xlp4clja
60.8
xlpaldpa62.6
xlpzba xlp4clja-277
73.0
xpps0433a xpps0433c
80.0
xpps0003a
81.7
xlpchie
83.8
xpps0040b
85.0
xlpffta.1
91.1
xpps0453c97.7
xlptht
100.7
xlpssrk09f06.120
108.4
xpps0106a
120.4
xlpssrk02d08.b157.4
BMC Plant Biology 2009, 9:62 />Page 14 of 22
(page number not for citation purposes)
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 prov-
enance.
Comparative genomics analysis of perennial ryegrass R
genes
Comparative analysis of R gene SNP loci and correspond-
ing ortholoci confirmed previously reported macrosyn-
tenic 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 pro-
vides the basis for testing R gene functionality in response
to a broader range of plant diseases, requiring significant
improvements of pathogen phenotyping [74] and corre-
sponding 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 diver-
sity and putative evolutionary origins. Efficient in vitro dis-
covery methods allowed assignment of R gene-derived
SNPs to genomic locations, revealing coincidence with
pathogen resistance QTLs in ryegrasses, as well as compar-
ative 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
) crossFigure 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.
xlprg48-200ct
0.0
xpps0111a
7.8
xlphbb-120ca12.2
xlphbb xlpinva
13.2
xlprg28-340cg
27.0
xlpssrh10g02
57.7
xlpssrh07g05
63.4
xlptlb64.2
xlprg26-298gt
65.1
xlprg27-743ct
xlprg27-912ag
68.4
xlpssrk02c09.1 xlpssrk14c12
71.4
xlpssrk05a11.2
72.2
xpps0289b xlpssrh01h01
72.8
xpps0195b74.7
xpps0273a
77.5
xlpssrk11g10

79.4
xlpdefc
82.1
xlppih
83.8
xpps0169a
85.5
xpps0074a
89.5
xlpssrk03b03.194.2
xlpmads3.1
99.1
xpps0036b
102.3
xlpsaltb
106.7
xlpdeff
108.9
xlpgluck-394ct113.9
xlpgluck
115.3
xpps0397a
121.4
xlpesi3b
144.3
xlpesi3g
152.1
AU
6
-LG5

AU
6
-LG7
AU
6
-LG6
xlpssrk01c04
0.0
xpps0098a
8.8
xpps0020b xpps0210a14.2
xlpssrh08h05.1
17.2
xlpomt2
19.5
xlpssrk05h01 xlphbd
xlpspsf
21.5
xlpcwinv-136tg
24.7
xlpinva-304ga xlpcwinv2
25.5
xlpp alb26.6
xlpssrh02h05.1
28.3
xlpd efa
29.9
xlpssrk11g12
32.8
xlpssrh02h05

37.6
xlpssrk13h08 xlpssrh05g07
42.3
xpps0192a
43.0
xpps0374a44.0
xpps0022b
46.0
xpps0310a
47.5
xpps0450b
48.3
xlpssrk09c10
51.8
xpps0241a55.5
xlpffta.2
56.7
xlpssrk10b07
63.3
xpps0019a
65.7
xpps0187a
78.9
xlpcat-353gt
86.5
xlpdfrb
92.6
xpps0299a100.4
xlpccra
108.1

xlprg47-625gt
137.7
xlpzbb
0.0
xlpssrh03a08.2
10.8
xpps0312a15.2
xlpssrk14f07
19.3
xlpa22a.2
21.5
xlpthc-148at
22.6
xlpssrk12h01.4
26.3
xlpsucsyn
33.5
xlpcia xlpcia-101at
34.5
xpps0411c35.8
xlpssrk08a06.1
37.8
xlpleaa
39.7
xpps0049c
44.1
xlprg60-216gt
45.9
xpps0376a48.7
xpps0441b

49.5
xpps0411d
50.5
xpps0466a
51.5
xlpssrk05b11
53.1
xlpccha-326cg xlpomt3
55.8
xpps0438b xpps0099b
60.5
xpps0424b61.2
xlpcadlike05-27.3ca
69.7
xlpccha
77.0
xlpcysa
84.0
xlpthc.2 xlpthc.1
90.4
xlpccrb
105.7
BMC Plant Biology 2009, 9:62 />Page 15 of 22
(page number not for citation purposes)
Genetic linkage maps of LGs 1,2 and 5 from the p150/112 reference populationFigure 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 indi-
cated 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.

p150/112 - LG2
p150/112 - LG5
xlpssrh03a08
0.0
xcdo38.1 e35t59220
e33t62225
13.5
e41t5023115.9
xcdo405
22.9
xlpssrh02d10.1
26.4
xlpssrh09e12.1
27.9
e33t62515
29.4
e41t4722532.2
xlprg13-380ag xlprg40-31cg
32.5
xlpssrh03f03 xlpssrk13c10
xlpssrk14b06 xlpssrh01a07
35.3
xcdo385.2
36.0
xlpssrk09f06.1 xlpssrk02e02
e35t59112
36.7
e33t5013338.2
e33t62460
39.7

xlpssrk12e03
41.2
xpsr901 e36t48595
42.7
e33t62113
44.4
e35t59575
49.8
e41t50240 xcdo1417
xlprg50-464ca
55.2
xlpssrh08h05.3
58.7
xlprg24-460at
62.9
xc600a
77.9
e33t62620
86.4
xpsr540b88.1
e40t50334
96.3
xlpssrk08f05
102.0
xlpssrk12e06 xlpssrhxx285
xcdo36 e40t49173
111.1
xc472
114.7
xr738 xc556

116.5
xc847129.3
e36t50375
0.0
xlpssrk03f09
13.1
xlpssrh11g05
15.4
xlpssrh02e12 e41t47750
18.7
xlprg27-912ag
19.2
xlprg27-743ag
19.7
xlprg26-298gt
20.2
xlpssrk09c10
20.7
e33t50112
23.5
e41t50590
28.9
xlpssrh10g02 e41t47198
xlpssrh07g05 xlpssrk14c12
30.6
xlpssrk05h02
33.8
e41t47445 xlpssrk15a07
Xablpg26x
37.3

xcdo412
43.6
orsb xlpssrk03b03
e38t50311
47.1
xr1751
49.6
e33t62210
52.2
xlpgluck-394ct
e38t50189
63.1
e33t50147
74.0
e33t62101
75.7
xr2710 e41t5020082.7
e40t50268 xcdo400
86.0
xrz404
101.0
p150/112 - LG1
e33t508000.0
xc1239Aa
8.9
e41t47500
12.7
e33t62180 xcdo580
22.6
xlpssrk10f08

25.6
xlpssrhxx238 xlpssrk14c04
xlpssrk15h05 xlpssrk09g05
xlpssrk03a02 xlpssrh02h04
xcdo98
28.5
xlpssrk07f07
29.2
xlpssrk12d11
29.9
xlpssrh09e12.2
30.6
xlpssrk14d0231.3
xlpssrh07g03
32.3
xlpssrh12g03
33.3
xpsr601 xcdo105a
xbcd1072a
36.2
xlpssrk10g04
41.8
xbcd738 xpsr162
e41t47180
45.0
e33t50175
56.6
xcdo202
89.7
e41t59188

93.5
xlpnox-1131at
122.6
e33t61133152.1
BMC Plant Biology 2009, 9:62 />Page 16 of 22
(page number not for citation purposes)
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 studiesFigure 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].
xlpap2b.2
0.0
xlprg1-369ct
34.7
xlpesi3f
56.9
xlpesi3e.1 xlplt16ab
66.8
xlpssa.2 xlplt16aa71.6
xlpa05_071Qs292
76.3
xlptrx-649ag xlpssrk12d11-038cg
79.3
xlpbcd762-274at
81.0
xlpcdo1173-212ac xlpzta-299ct
xlpssrk10f08
85.3
xlpsalta.2
89.7
xpps0136c93.9
xpps0094a
96.3
xpps0305a
97.9
xlpcadc-561.45tg
100.8

xlppsr168-699ag
101.5
xlpcdo98-596ac103.9
xlpf5h.2
108.8
xlpssrk07f07-103at
111.5
xlpcadc
114.4
xlpssrk07f07
115.7
xlpssrk15h05
117.4
xpps0066b
120.7
xlpssrk08a06.2122.8
xlpssrh12a04 xlpssrh11g05
128.7
xlppif
132.7
xlpssrh12g03
133.4
xlpthbna-317ag
135.0
xlposeindel
138.2
xlpwalie
141.4
xlpwalic-170ag xlpchihs.1142.5
xlp4cljb

143.2
xlpmtd
143.9
xlprg8-271ct
151.6
xlpssrk14c08
155.6
xlpwalic156.5
xlpssrk10g04
158.5
xpps0381a
159.5
xpps0174a xpps0055a
160.5
xpps0231a
162.5
xpps0038b
164.7
xlprg29-293ct
176.1
xlpnox-1131at176.4
xlpnox-735ag
176.7
xpps0211b xpps0114a
177.4
NA
6
-LG1
PC400-014
0.0

LPSSRK03A02_b
5.3
0SE_b13.7
PC407-045
14.4
Xrgc488_b
19.9
OSE_x
23.9
PC400-149
27.0
LPSSRK15H05
29.9
LPSSRK10F08
31.2
LPSSRK12D11_a32.7
rye023_c
33.7
PC026-03
35.5
rye023_a
36.1
PC400-032
36.3
LPSSRK03A02_a
36.9
LPSSRK07F07_b
41.0
LPSSRK10F08_g44.4
PC400-091

50.6
LPSSRK10G04
51.8
PC008-076
54.0
PC168-023
54.3
PC026-3259.7
PC008-085
61.7
PC026-34
67.5
PC026-R2
72.6
PC106-R2
78.8
PC168-R1
87.6
LpPc2
LpPc4
F
1
(SB2 x TC1)- LG1
BMC Plant Biology 2009, 9:62 />Page 17 of 22
(page number not for citation purposes)
Comparative mapping analysis between candidate R gene SNP loci mapped in the p150/112 population and QTLs for crown rust resistance from other published studiesFigure 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].
p150/112 - LG2
xlpssrh03a08
0.0
xcdo38.1 e35t59220
e33t62225
13.5
e41t5023115.9
xcdo405
22.9
xlpssrh02d10.1
26.4
xlpssrh09e12.1
27.9
e33t62515
29.4
e41t4722532.2
xlprg13-380ag xlprg40-31cgg
32.5
xlpssrh03f03 xlpssrk13c10
xlpssrk14b06 xlpssrh01a07
35.3
xcdo 385.2
36.0
xlpssrk09f06.1 xlpssrk02e02
e35t59112

36.7
e33t5013338.2
e33t62460
39.7
xlpssrk12e03
41.2
xpsr901 e36t48595
42.7
e33t62113
44.4
e35t59575
49.8
e41t50240 xcdo1417
xlprg50-464ca
55.2
xlpssrh08h05.3
58.7
xlprg24-460at
62.9
xc600a
77.9
e33t62620
86.4
xpsr540b88.1
e40t50334
96.3
xlpssrk08f05
102.0
xlpssrk12e06 xlpssrhxx285
xcdo36 e40t49173

111.1
xc472
114.7
xr738 xc556
116.5
xc847129.3
PC407-006
0.0
PC400-11310.8
rye024_c
14.2
PC407-055PC400-137
20.4
Xcdo385
22.9
LPSSRK14B06
24.6
PC026-R4
28.4
PC106-022
30.4
PC026-R331.9
PC078-121
33.0
PC026-39
34.6
rye024
36.5
Xcsu6
40.7

PC008-02843.6
PC001-032
44.9
PC400-075
46.6
PC400-140
50.7
PC001-079
51.3
PC078-057
52.2
PC400-036
54.1
LPSSRK09G12a56.5
Xcdo456
57.8
PC106-106
59.5
PC008-088
61.7
PC168-051
62.0
PC008-05067.1
PC400-077
68.0
PC078-075
68.1
rye022
70.0
PC400-022

70.5
PC078-099
75.4
Xbcd135
81.7
PC008-01484.9
LPSSRK08F05_c
88.8
LpPc1 LpPc3
F
1
(SB2 x TC1)- LG2
Pca
BMC Plant Biology 2009, 9:62 />Page 18 of 22
(page number not for citation purposes)
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 puta-
tive 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 DNA-
derived SSR markers (indicated in bold black italics) and an orange dotted line links the genetic map positions of LrK10
ortholoci.
Perennial ryegrass
NA
6
-LG1
Perennial ryegrass
p150/112 - LG1
e33t508000.0
xc1239Aa
8.9

e41t47500
12.7
xcdo580
22.6
xlpssrk10f08
25.6
xlpssrhxx238
xlpssrk15h05
xlpssrk03a02
xcdo 98
28.5
xlpssrk07f07
29.2
xlpssrk12d11
29.9
xlpssrh09e12.2
30.6
xlpssrk14d0231.3
xlpssrh07g03
32.3
xlpssrh12g03
33.3
xpsr601
xbcd1072a
36.2
xlpssrk10g04
41.8
xbcd738 xpsr162
e41t47180
45.0

e33t50175
56.6
xcdo202
89.7
e41t59188
93.5
xlpnox-1131at
122.6
e33t61133152.1
xlpap2b.20.0
xlprg1-369ct
34.7
xlpesi3f
56.9
xlpesi3e.1 xlplt16ab
66.8
xlpssa.2 xlplt16aa
71.6
xlpa05_071Qs292
76.3
xlptrx-649ag
79.3
xlpbcd762-274at81.0
xlpcdo1173-212ac
xlpssrk10f08
85.3
xlpsalta.2
89.7
xpps0136c
93.9

xpps0094a96.3
xpps0305a
97.9
xlpcadc-561.45tg
100.8
xlppsr168-699ag
101.5
xlpcdo98-596ac
103.9
xlpf5h.2
108.8
xlpssrk07f07-103at
111.5
xlpcadc114.4
xlpssrk07f07
115.7
xlpssrk15h05
117.4
xpps0066b
120.7
xlpssrk08a06.2
122.8
xlpssrh12a04
128.7
xlppif
132.7
xlpssrh12g03133.4
xlpthb-317ag
135.0
xlposeindel

138.2
xlpwalie
141.4
xlpwalic-170ag
142.5
xlp4cljb143.2
xlpmtd
143.9
xlprg8-271ct
151.6
xlpssrk14c08
155.6
xlpwalic
156.5
xlpssrk10g04
158.5
xpps0381a
159.5
xpps0174a160.5
xpps0231a
162.5
xpps0038b
164.7
xlprg29-293ct
176.1
xlpnox-1131at
176.4
xlpnox-735ag
176.7
xpps0211b

177.4
cdo580-2(5)
0.0
LrK10
8.0
E32/M-CAGC185
17.0
E32/M-ctag307
27.0
P347(10)
35.0
E35/M-CAAC47
47.0
E32/M-CACT186
64.0
E35/M-CTTC57
84.0
16-06G269
115.0
Italian ryegrass
-LG1
avnA
0.0
bcd1482b
11.0
LrK10
15.0
cd o1423a
21.0
cd o718b

29.0
cd o580
38.0
43.0
cdo1173a
49.0
re4m2_7x
56.0
pta71a
84.0
cdo187
94.0
Hexaploid oat
LG 4_12
xHor2
0.0
xLrK10-A
4.4
xMla5.1
xMla6
8.0
xmwg837
8.6
xHo21
10.2
Barley - 1HS
xmwg938
5.0
xmwg206
6.0

XpLrk10-A
6.5
Xmwg837
12.2
Rye - 1RS
xLrK10
x(Glu-3)1A
Wheat - 1AS
5.7
BMC Plant Biology 2009, 9:62 />Page 19 of 22
(page number not for citation purposes)
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-concep-
tualised 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 devel-
oped and contributed genetically-defined plant materials.
GS provided EST sequence information and valuable edi-
torial advice. JF co-conceptualised the project, provided
overall project leadership, and co-developed interim and
final drafts of the manuscript.
Additional material
Additional File 1

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
[ />2229-9-62-S1.doc]
Additional File 2
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 data-
bases and employs known gene ontology and Pfam domain analysis to
assign putative function to candidate sequences. Nomenclature of paralo-
gous sequences is based on the unique identifier for the primary template
sequence (e.g., LpESTe11_14) followed by a numerical suffix (.1, .2
etc.), e.g. LpESTe11_14rg.1.
Click here for file
[ />2229-9-62-S2.doc]
Additional File 3
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
[ />2229-9-62-S3.doc]
Additional File 4
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
[ />2229-9-62-S4.doc]
Additional File 5
Major protein sequence motifs in predicted Lolium NBS domains.
a
Motifs 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];
b
Bioinformatic 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);
c
Consensus amino acid sequences for
Lolium NBS sequences were derived from MEME, while those for wheat
were derived from [58].
Click here for file
[ />2229-9-62-S5.doc]
Additional File 6
Summary information of amino acid structure for NBS domain pro-
tein sequence motifs numbered in Fig. 2, based on matching using the
MEME program.
Click here for file
[ />2229-9-62-S6.doc]
Additional File 7
Reference information for sequences corresponding to individual clus-

ters identified during phylogenetic analysis for the complete NBS
domain (P-Loop-GLPL), as depicted in Additional File 8.
Click here for file
[ />2229-9-62-S7.doc]
Additional File 8
NJ dendrograms based on amino acid alignment of the full-length (P-
Loop – 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
[ />2229-9-62-S8.ppt]
Additional File 9
Reference information for sequences corresponding to individual clus-
ters identified during phylogenetic analysis for the Kin-2A-GLPL
region of the NBS domain, as depicted in Additional File 10.
Click here for file
[ />2229-9-62-S9.doc]
BMC Plant Biology 2009, 9:62 />Page 20 of 22
(page number not for citation purposes)
Acknowledgements
This work was supported by funding from the Victorian Department of Pri-
mary Industries, Dairy Australia Ltd., the Geoffrey Gardiner Dairy Founda-
tion, Meat and Livestock Australia Ltd. and the Molecular Plant Breeding
Cooperative Research Centre (MPB CRC).
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Additional File 11
Summary information for LAP and SNuPe primers used for predicted
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Comparative chromosomal positions of predicted putative orthologous
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Click here for file
[ />2229-9-62-S12.ppt]
Additional File 13
Comparative chromosomal positions of predicted putative orthologous
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xlprg54-688ag) on NA
6
-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
[ />2229-9-62-S13.ppt]
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