BACKGROUND
plants have evolved complex mechanisms to defend against pathogen attack.
two types of immunity have been described: pathogen-associated molecular pattern -triggered immunity  and effector-triggered immunity .
pti is induced at an early stage when pamps are recognized by pattern recognition receptors , whereas eti is induced by direct or indirect association of a resistance  protein with a pathogen-derived effector  <cit> .
the outcomes of the two immune systems appear to be partial or quantitative resistance and non-host resistance , and qualitative resistance .
recently, niks and marcel  <cit>  proposed that the varying efficacy of pti suppression by pathogen effectors may explain partial resistance.
in cereal crops, the barley-puccinia hordei otth  pathosystem is a model for investigating partial and non-host resistance.
microscopic studies on resistance levels in relation to the pathogen developmental phases has indicated plant cell wall penetration and haustorium formation by p. hordei as critical phases determining the success or failure of the infection  <cit> .
pre-haustorial resistance reduces the chance of successful haustorium formation by the fungal pathogen in the host cells.
failed attempts are typically associated with cell wall appositions  <cit> .
such pre-haustorial basal host defence is a typical reaction to ph-infection in most  barley lines exhibiting partial resistance  <cit> .
post-haustorial resistance is usually due to r gene-mediated hypersensitive response after haustorium formation  <cit> .
these two types of resistance have strategic significance in plant breeding for resistance to diseases.
quantitative or partial resistance has become increasingly important because of its broader spectrum and higher durability compared to r-gene mediated race-specific resistance.
many of the genes underlying partial resistance have plant developmental stage-dependent effectiveness  <cit> .
currently, over  <dig> quantitative trait loci  for quantitative basal resistance to leaf rust from five different mapping populations have been mapped to barley chromosomes  <cit> .
they are named rphq genes .
of these,  <dig> were effective during the seedling stage, and were detected by qtl analysis of the latency period exhibited by the rust fungus on seedling leaves  <cit> .
considerable effort has been expended in an attempt to identify the genes underlying these qtl.
notably, a set of nils and reciprocal nils have been developed that contain single  or combined  introgressed segment carrying resistance and susceptibility qtl allele that were identified in an l <dig> × vada ril population  <cit> .
l <dig> is an ethiopian landrace and highly susceptible to barley leaf rust.
vada is a dutch cultivar expressing a high level of partial resistance.
following a positional cloning strategy, marcel et al.
<cit>  have fine-mapped rphq <dig>  the qtl with largest effect, to an interval of  <dig>  cm corresponding to less than  <dig> kb in physical length.
microarray technology is being widely used to address various biological, biochemical and genetic questions.
microarray-based gene expression studies can be generally grouped into two major categories.
the first aims to address specific biological questions by monitoring the differential expression of genes under contrasting conditions or over time.
the most common studies in this field are the investigations on host-pathogen interactions.
profiling changes in genome-wide expression in response to pathogen challenge has identified a large spectrum of genes that are responsive to pathogen attack or are associated with plant resistance in various pathosystems .
the second category is based on the more recently emerged concept of 'genetical genomics'  <cit>  or expression qtl  mapping that combines highly parallel gene expression studies with the power of genetic segregation.
eqtl studies have been performed on maize, eucalyptus and arabidopsis  <cit> .
eqtl analyses in barley have addressed the global genetic architecture of transcript abundance in
<cit> , the phenomenon of limited pleiotropy  <cit>  and as an approach to identify the causal or candidate genes underlying partial resistance to fungal diseases  <cit> .
while both categories of microarray studies are based on variation in transcript abundance, eqtl analysis provides a genetic dimension that can differentiate cis-from trans-regulation and the genetic locations of a large number of genes through the co-location of high lod eqtl  and their structural genes  <cit> .
this is particularly valuable for a crop with large and unsequenced genome like barley.
here, using a previously reported agilent  <dig> k custom array  <cit> , we performed differential expression analysis of qtl-nils and their recurrent parental lines at  <dig> hours post-inoculation  with puccinia hordei.
our major objective was to identify candidate genes for rphq <dig> and rphq <dig>  in addition, transcript profiles between ph-infected parents and their respective mock-inoculated controls allowed the establishment of transcriptomic signatures for each line in response to ph-infection.
our results indicate that transcriptional differentiation between qtl-nils and their respective recurrent parents reveals components of a regulatory transcriptional relay induced in response to ph-infection.
the datasets generated offer a basis for further studies on defence signalling in relation to partial resistance to p. hordei in barley.
RESULTS
transcriptomic signatures of response to p. hordei
plant defence responses involve transcriptional activation of a plethora of specific genes and regulation of their temporal and spatial expression  <cit> .
to investigate the genome-wide transcriptional signatures of susceptible and partially resistant barley lines l <dig> and vada respectively in response to p. hordei infection, we compared ph-infected with mock-inoculated leaf material.
a stringent threshold with fold change > <dig> and false discovery rate  < <dig>  was adopted for declaring significant differences.
at this threshold,  <dig> and  <dig> genes were respectively identified in l <dig> and vada as 'significantly differentially expressed' with  <dig>  overlapping between the two lines while  <dig>  were present in only one of the two parents .
this yielded a total of  <dig> genes which we considered 'ph-responsive'.
close examination of the expression data of the  <dig> 'ph-responsive' genes from both parents showed that while a substantial number failed to meet the stringent thresholds applied  they still exhibited statistically significant differential expression in both parents.
therefore, a relaxed threshold ignoring the fold changes was adopted for the follow-up analysis on the commonality and specificity of response to ph-infection between the resistant and susceptible lines using all  <dig> ph-responsive genes.
we plotted the log-transformed expression ratios of vada against l <dig> and classified them into four groups.
genes that showed the same expression patterns  and expression changes at p <  <dig>  in both lines were defined as being common to both lines , whereas, those that showed significant expression changes in one line but no significant expression changes  or a contrasting expression pattern in the other line were considered as being line-specific .
the remaining genes that had no strong evidence to suggest either commonality or specificity were grouped into 'not determined' .
there were a total of  <dig>   <dig>   <dig> and  <dig> genes that appeared to be in common, vada- or l94-specific or 'not determined' representing 73%, 3%, 4% and 20% of the  <dig> ph-responsive genes respectively.
figure  <dig> shows a colour-coded heat map that was converted from the relative expression ratios  of the  <dig> genes and the  <dig>  line-specific genes showing the overall similarity and specificity of gene expression in l <dig> and vada.
full expression information of the  <dig> genes and the line-specific genes is given in the additional file  <dig>  and  <dig>  respectively.
genome-wide ph-responsive genes have previously been investigated  <cit>  using steptoe  and morex , two barley cultivars with similar, intermediate levels of resistance to p. hordei .
we therefore compared the data from l94/vada with those from st/mx.
at exactly the same thresholds  a total of  <dig> genes were identified as ph-responsive in st/mx  <cit> .
applying exactly the same criteria as described above, we identified  <dig> ,  <dig> , and  <dig>  genes that were common, st-specific and mx-specific respectively.
we then explored the common genes in each of these categories between the two experiments .
<dig> %  of the  <dig> genes detected with l94/vada were also detected with st/mx and more than half of the genes  were significant in all four lines, highlighting the similarity of response to ph-infection across genotypes.
of the  <dig> and  <dig> genes that were specifically detected in vada and l <dig> respectively,  <dig> and six of these were reproducibly identified as ph-responsive in steptoe or morex .
all of the  <dig> vada -specific genes were up-regulated in vada and st or mx.
ten of these genes showed significant differential expression  between st and mx.
of the six l <dig> -specific genes, only one up-regulated gene  showed significant differential expression between st and mx .
note: ph-responsive genes were selected on criteria with fold change > <dig> and fdr < <dig> ; genes with similar patterns were selected from ph-responsive gene on p <  <dig>  without considering fold changes.
to further characterise the biological processes represented by the  <dig> ph-responsive genes, we performed gene ontology  analysis by classifying the ph-responsive genes into functional biological categories based on go terms retrieved from their rice homologues through the rice database at http://rice.plantbiology.msu.edu/annotation_pseudo_goslim.shtml.
the ph-responsive genes were associated with a broad range of biological processes.
the primary category was related to defence response.
we further classified these genes into  <dig> major functional categories following the go terms in 'biological process' with all remaining genes grouped into 'other functions' or 'unknown'.
the results are shown in figure  <dig>  they indicate that at the sampling time point of  <dig> hpi, the plants had responded to defend against the ph-infection.
differential expression analysis between ph-infected recurrent parents
we performed genome-wide differential expression analysis by comparing expression differences between ph-infected l <dig> and ph-infected vada at  <dig> hpi.
a total of  <dig> genes were identified as being differentially expressed , of which  <dig> were ph-responsive genes as described above.
the majority  represent genome-wide, genotype-specific differences in gene expression.
the detailed information of these genes regarding their expression ratios, p-values and functional annotations is presented in additional file  <dig> .
differential expression between ph-infected qtl-nils and ph-infected recurrent parents
to identify qtl-specific and differentially expressed genes accounted for by genetic differences in the qtl regions, the two reciprocal qtl-nils were compared with their respective recurrent parents: l <dig> vs. l94-rphq2+ <dig> and vada vs. vada-rphq2+ <dig>  a total of  <dig> genes were identified as significant  in at least one comparison.
of these,  <dig> genes showed a significant difference in one recurrent parent/qtl-nil comparison but not with the other.
we attribute these observations to the different size and incomplete overlap of the introgressed segments in the two recurrent parent/qtl-nil pairs.
these genes were, therefore, not pursued further.
the remaining  <dig> genes showed expression differences at p <  <dig>  in both comparisons and were, therefore, considered potentially relevant to the qtl regions.
this suggests that differential expression results from genetic factors differing specifically within the qtl regions .
of these  <dig> genes,  <dig> were present on the list of  <dig> differentially expressed genes between ph-infected vada and ph-infected l <dig>  the remaining five genes  did not fulfil the criteria  set for the differential expression between the two ph-infected recurrent parents, but their expression differences were still statistically significant .
transcription of qtl-specific and differentially expressed genes in response to ph-infection
to identify whether the  <dig> qtl-specific and differentially expressed genes were also ph-responsive, expression data from the ph-infected vs. mock-inoculated experiment was re-investigated .
six genes showed changes that fulfilled the criteria  set for defining ph-responsive genes.
twelve genes did not fully meet the criteria, but their level of differential expression was still statistically significant  in at least one of the lines.
the others were not statistically significant.
identification of positional candidates for rphq <dig> and rphq3
to determine the map position of the  <dig> qtl-specific and differentially expressed genes, we took advantage of available datasets previously generated in three different eqtl studies  and ph-infected seedling leaves  <cit> .
<dig> of the  <dig> genes had one or more eqtl in at least one of these three experiments, yielding  <dig> eqtl in total.
the distribution of these eqtl was investigated by plotting their map positions against their lod/lrs values .
<dig> genes with eqtl mapped to within the qtl regions  , of which  <dig>  had lod > <dig> or lrs > <dig> suggesting they are cis-eqtl .
we then explored three available gene-based mapping datasets: illumina opa-snps  <cit> , single feature polymorphisms  and tdms  <cit>  to help assign genetic map positions to the  <dig> genes.
this allowed four and nine genes to be placed within the confidence intervals of rphq <dig> and rphq <dig> respectively.
all of these genes overlapped with the eqtl except two  for which no eqtl was detected in the three eqtl studies .
rphq <dig> and rphq <dig> on chromosome 2h and 6h are syntenic to regions on rice chromosomes os <dig> and os <dig> respectively.
conservation of synteny allowed us to infer the approximate map positions of an additional  <dig> genes to within the qtl regions .
thus, of the  <dig> qtl-specific differentially expressed genes, the map location of nine and  <dig> fell within rphq <dig> and rphq <dig> respectively, whilst the  <dig> others remain unknown.
of note was the observation that one gene , encoding an ethylene-responsive transcription factor  <dig>  , has been mapped as illumina opa-snp marker 11_ <dig> to chromosome 1h at position  <dig> cm  <cit> .
this map position is consistent with a location based on conservation of synteny between rice os <dig> and barley chromosome 1h, suggesting that differential expression of this gene is the consequence of trans-regulation by a gene located within either rphq <dig> or rphq <dig>
discussion
in this study, we performed differential expression analysis of two reciprocal qtl-nils and compared them with their respective recurrent parents.
as qtl-nils differ genetically from their recurrent parent only in the selected qtl regions, we would anticipate that genetic polymorphism between these qtl regions would account for any differential expression observed.
however, due to the complexity of gene regulation, differentially expressed genes may not necessarily be located in the introgressed qtl regions, which may themselves contain regulatory genes affecting the expression of other genes spread throughout the genome.
we therefore established the map positions of differentially expressed genes by exploiting previously generated gene mapping datasets.
of the  <dig> genes highlighted in our comparisons between nils and recurrent parents,  <dig> detected eqtl in at least one of the three previous eqtl studies and co-located at the qtl regions, most  having high lod/lrs scores  <cit>  .
eqtl with high lod scores have been demonstrated previously to be almost always cis-eqtl  <cit>  placing these genes within the rphq <dig> or rphq <dig> qtl regions.
the observation that so many significantly differentially expressed genes appeared to be regulated in cis- is in agreement with previous studies  <cit> .
an exception was unigene  <dig>  encoding hverf <dig>  this gene mapped to  <dig> cm on chromosome 1h, consistent with the position of its rice homologue os05g <dig>  predicted by conservation of synteny  <cit> .
this observation raises the possibility that the introgressed regions at either rphq <dig> or rphq <dig> contain a polymorphic trans-acting regulator that differentially modulates expression of hverf <dig>  no eqtl for hverf <dig> was detected at the regions corresponding to rphq <dig> or rphq <dig> in the st/mx dh mapping population, consistent with the fact that it does not segregate for rphq <dig> or rphq <dig>
hverf <dig> is a member of a family of plant transcription factors functionally involved in defence signalling pathways related to ethylene, jasmonic acid and abscisic acid.
over-expression of arabidopsis aterf <dig> represses the expression of pathogenesis-related  genes such as basic chitinase and beta- <dig> -glucanase genes and genes containing a gcc-box  <cit> , the core sequence element of promoters required for responsiveness to ethylene  <cit> .
in our previous experiment with steptoe and morex, cultivars with similar but intermediate levels of partial resistance to leaf rust, we also observed that hverf <dig> was significantly up-regulated by ph-infection but no differential expression  was observed between the parents  <cit> .
here, hverf <dig> was induced in ph-infected l <dig>   and vada   as compared to mock-inoculated controls , and the expression level of the vada allele was only a third  of that of the l <dig> allele after induction.
the association of resistance/susceptibility with lower/higher expression of hverf <dig> appears to be in agreement with the negative regulatory role of hverf <dig> on the expression of pr and other defence responsive genes.
however, consistent association of higher expression of pr genes with resistance was not observed in vada and l <dig>  this may reflect the general complexity of natural resistance response coupled with allelic variation at pr genes between these two lines.
while this train of inference highlights hverf <dig> as potentially important in this specific defense interaction, none of the so far reported  <dig> qtl for partial resistance to leaf rust, nor any of the qtl for resistance to heterologous rusts is co-located with hverf <dig> at  <dig> cm on chromosome 1h  <cit> .
thus, hverf <dig> is not a positional candidate for any of the reported qtl.
however, of direct relevance is a previously highlighted eqtl hotspot for
genes that was associated with opa-snp 11_ <dig>  <cit>  at  <dig> cm  on chromosome 1h spanning the region containing hverf <dig>  this hotspot comprised  <dig> eqtl in less than a  <dig> cm interval and contained genes primarily involved in defence response  <cit> .
given its known role in pr-protein regulation, we speculate that hverf <dig> represents a key regulatory relay component of the signalling pathway that controls expression of at least a portion of the genes with eqtl located at the hotspot on chromosome 1h.
considering these observations together we hypothesise that the causal genetic polymorphism at either rphq <dig> or rphq <dig> differentially regulates hverf <dig> in trans , the consequence of which is differential regulation of down-stream defence responses.
in this scenario, the candidate genes for rphq <dig> or rphq <dig> would be those acting up-stream rather than down-stream of hverf <dig> and possibly involved directly or indirectly in plant hormone signalling pathways.
while we did not find such a candidate from the annotated functions of the qtl-specific and differentially expressed candidates for rphq <dig> and rphq <dig>  the gene controlling expression of hverf <dig> may, however, not be differentially expressed between l <dig> and vada, may not be on our expression platform  or may not be at the orthologous position in rice.
an alternative to identifying the causal gene for rphq <dig> or rphq <dig> could be through map-based cloning of the trans-eqtl for hverf <dig>
marcel et al.
<cit>  narrowed down the genetic interval for rphq <dig> to  <dig>  cm corresponding to a physical length of  <dig> kb in barley  and a  <dig>  kb syntenic region on rice chromosome  <dig>  inspection of all predicted genes in the rphq <dig> syntenic interval in rice identified a cluster of six peroxidase genes and a map3k gene  <cit>  as potential candidates because of their functional involvement in defence responses.
in this study, we identified four barley genes at rphq <dig> that were differentially expressed and had homologues located in the syntenic region in rice , unigene <dig> , unigene <dig>  and unigene <dig> ).
unigene <dig> and  <dig> both encode peroxidases and are within the  <dig>  cm interval containing rphq <dig>  the other two, according to the fine mapping data of marcel et al.
<cit>  fell just outside the candidate interval.
however, given the frequent breakdown in conservation of synteny, positional candidate gene identification using this approach alone remains problematic.
differential expression in the qtl-nils identified an additional five candidate genes  that were not apparently present in the syntenic region of rice.
two of these encode proteins that are functionally involved in signal transduction , one pr protein, and one with homology to human nipa <dig>  implicated in prader-willi/angelman syndrome  <dig>  <cit> .
one gene showed no homology to known genes.
thus, these five genes, together with the two peroxidase genes, are potential positional candidates for rphq <dig>  further refinement of the candidate gene list will require knowledge of the role of these genes in defence response and correlation of transcript levels with resistance/susceptibility.
many more genes  were identified as being differentially expressed and located at the rphq <dig> region.
this is expected given the larger interval of the qtl  and rphq <dig> may, therefore, account for differential expression of most of the genes with unassigned map positions.
functionally, none of the differentially expressed genes at rphq <dig> or rphq <dig> appear to be obvious candidates for a regulator of hverf <dig>
many defence genes encoding pr proteins and components of the phenylpropanoid pathway such as phenylalanine ammonia lyase  were, as would be expected, ph-responsive.
pr genes encoding beta- <dig> -glucanases, chitinases and thaumatin-like proteins exhibit strong in vitro anti-fungal activity  <cit>  and numerous studies have shown that transgenic plants expressing pr-proteins have significant improvement of disease resistance  <cit> .
pal, the first committed enzyme in the phenylpropanoid pathway, is involved in synthesis of both phytoalexins and lignin.
phytoalexins are antimicrobial while lignin synthesis contributes to formation of papillae, which are physical barriers against cell wall penetration by the pathogen  <cit> .
as part of the general response to pathogen infection, few of the genes fell into these categories co-located at the two qtl for partial resistance.
one exception, unigene <dig>  encoding a chitinase , co-located at rphq <dig>  was ph-responsive and differentially expressed between rphq <dig> and rphq <dig>  however, it was not prioritized as a candidate for rphq <dig> since the higher level of gene expression was associated with the susceptibility allele rphq <dig>  whereas a number of defence genes were activated in response to ph-infection, none was found to be a promising candidate for rphq <dig> or rphq <dig>  our results support the notion that components of the general defence response have incremental, rather than deterministic, roles in the outcome of an interaction between a plant and a pathogen  <cit> .
many attempts to identify genes for disease resistance have highlighted those involved in signal transduction  <cit>  or physiological and cellular functions  <cit>  rather than defence per se  <cit> .
ph-infection triggers a broad range of biological responses with defence response genes being significantly over-represented.
of note is a set of genes encoding receptor-like kinase , receptor-like proteins , wrky, mapk and pr proteins , which form a complete and well-explored defence signalling cascade starting with the perception of pamps, activation of wrky transcription factors and the subsequent induction of pr proteins  <cit> .
our results also suggest that, in the absence of cognate r genes to p. hordei, plants still mount reactions similar to r-gene mediated responses as indicated by the significant up-regulation of genes coding for r gene  proteins and marker genes for oxidative burst such glutathione s-transferase and peroxidase.
although no obvious r genes were identified as candidates for the qtl in this study, r gene-like mediated responses may contribute to basal resistance as a complementary mechanism to pamp-triggered defence responses.
support for this is provided by observations that resistance qtl are often coincident with the location of r-gene homologues  <cit>  and that mutated r genes can induce a resistance phenotype similar to quantitative resistance controlled by multiple genes  <cit> .
one striking characteristic of the responses to ph-infection was the activation of signalling pathways related to a broad range of plant hormones including ethylene, gibberellins, auxin, and brassinosteroid as indicated by the up-regulation of genes encoding ethylene-responsive transcription factors, acc oxidase, auxin-responsive proteins, brassinosteroid insensitive 1-associated receptor kinase  <dig> , gibberellin receptors and a della protein .
all of these hormones have been reported to be involved in plant defence responses  <cit>  and various defence pathways are interconnected through hormone-mediated signalling pathways forming complex regulatory networks  <cit> .
here, the identification of the ethylene-responsive factor hverf <dig> as a putative link between pathogen perception and response is consistent with a role for differential hormone signalling in partial resistance.
understanding the role of rphq <dig> of rphq <dig> in initiating and coordinating the response requires further work.
substantial overlap of ph-responsive genes was identified in super-susceptible  and partially-resistant  lines.
over 70% of ph-responsive genes were detected in both l <dig> and vada and had the same expression patterns  in both lines.
an even higher percentage of overlapping ph-responsive genes  was discovered in both steptoe and morex, two cultivars with similar and intermediate level of partial resistance.
given that these lines are genetically diverse, we conclude that barley lines without known cognate r genes to p. hordei exhibit similar responses at the transcriptional level, and that observed differences are largely quantitative.
similar findings have been observed in the comparison between compatible and incompatible interactions  <cit> .
a small proportion  of ph-responsive genes in this study did appear to be resistant/susceptible line-specific and it may be that they determine part of the observed phenotypic differences between lines.
however, in ph-infected leaves we found no evidence for their differential expression in the comparisons between the two qtl-nils and their respective recurrent parents.
therefore, if the variation in resistance, accounted for by rphq <dig> or rphq <dig>  is regulated at the transcriptional level, these are not strong candidate genes.
we generated a robust expression data set in reciprocal rphq2/rphq <dig> qtl-nils at  <dig> hpi, which is the timepoint previously described as being the most critical during p. hordei invasion in barley  <cit> .
however, we realise that transcriptional re-programming in response to pathogen infection is a dynamic and complex process and that defence-associated genes respond to input stimuli with different timing and amplitude.
a limitation of our experiment is, therefore, that defence response scenarios constructed on the transcriptional profiles of the  <dig> ph-responsive genes identified here is simply a snapshot of a dynamic process, at the point when infection hyphae have just attempted penetration of the host cells forming haustoria  <cit> .
to extend our understanding of the complex regulatory mechanisms occurring during defence against p. hordei, a more comprehensive investigation would involve sampling at multiple timepoints covering the whole infection period.
CONCLUSIONS
differential expression with qtl-nils identifies genes predominantly located at the target region providing both transcriptional and positional candidate genes underlying the qtl.
positional analysis of the differentially expressed genes relative to the qtl has the potential to discover regulatory relays initiated from genes within the qtl.

 plants have evolved complex mechanisms to defend against pathogen attack. two types of immunity have been described: pathogen-associated molecular pattern -triggered immunity and effector-triggered immunity. pti is induced at an early stage when pamps are recognized by pattern recognition receptors, whereas eti is induced by direct or indirect association of a resistance protein with a pathogen-derived effector. the outcomes of the two immune systems appear to be partial or quantitative resistance and non-host resistance, and qualitative resistance. recently, niks and marcel proposed that the varying efficacy of pti suppression by pathogen effectors may explain partial resistance. in cereal crops, the barley-puccinia hordei otth pathosystem is a model for investigating partial and non-host resistance. microscopic studies on resistance levels in relation to the pathogen developmental phases has indicated plant cell wall penetration and haustorium formation by p. hordei as critical phases determining the success or failure of the infection. pre-haustorial resistance reduces the chance of successful haustorium formation by the fungal pathogen in the host cells. failed attempts are typically associated with cell wall appositions. such pre-haustorial basal host defence is a typical reaction to ph-infection in most barley lines exhibiting partial resistance. post-haustorial resistance is usually due to r gene-mediated hypersensitive response after haustorium formation. these two types of resistance have strategic significance in plant breeding for resistance to diseases. quantitative or partial resistance has become increasingly important because of its broader spectrum and higher durability compared to r-gene mediated race-specific resistance. many of the genes underlying partial resistance have plant developmental stage-dependent effectiveness. currently, over quantitative trait loci for quantitative basal resistance to leaf rust from five different mapping populations have been mapped to barley chromosomes. they are named rphq genes. of these, were effective during the seedling stage, and were detected by qtl analysis of the latency period exhibited by the rust fungus on seedling leaves. considerable effort has been expended in an attempt to identify the genes underlying these qtl. notably, a set of nils and reciprocal nils have been developed that contain single or combined introgressed segment carrying resistance and susceptibility qtl allele that were identified in an l × vada ril population. l is an ethiopian landrace and highly susceptible to barley leaf rust. vada is a dutch cultivar expressing a high level of partial resistance. following a positional cloning strategy, marcel et al. have fine-mapped rphq the qtl with largest effect, to an interval of cm corresponding to less than kb in physical length. microarray technology is being widely used to address various biological, biochemical and genetic questions. microarray-based gene expression studies can be generally grouped into two major categories. the first aims to address specific biological questions by monitoring the differential expression of genes under contrasting conditions or over time. the most common studies in this field are the investigations on host-pathogen interactions. profiling changes in genome-wide expression in response to pathogen challenge has identified a large spectrum of genes that are responsive to pathogen attack or are associated with plant resistance in various pathosystems. the second category is based on the more recently emerged concept of'genetical genomics' or expression qtl mapping that combines highly parallel gene expression studies with the power of genetic segregation. eqtl studies have been performed on maize, eucalyptus and arabidopsis. eqtl analyses in barley have addressed the global genetic architecture of transcript abundance in, the phenomenon of limited pleiotropy and as an approach to identify the causal or candidate genes underlying partial resistance to fungal diseases. while both categories of microarray studies are based on variation in transcript abundance, eqtl analysis provides a genetic dimension that can differentiate cis-from trans-regulation and the genetic locations of a large number of genes through the co-location of high lod eqtl and their structural genes. this is particularly valuable for a crop with large and unsequenced genome like barley. here, using a previously reported agilent k custom array, we performed differential expression analysis of qtl-nils and their recurrent parental lines at hours post-inoculation with puccinia hordei. our major objective was to identify candidate genes for rphq and rphq in addition, transcript profiles between ph-infected parents and their respective mock-inoculated controls allowed the establishment of transcriptomic signatures for each line in response to ph-infection. our results indicate that transcriptional differentiation between qtl-nils and their respective recurrent parents reveals components of a regulatory transcriptional relay induced in response to ph-infection. the datasets generated offer a basis for further studies on defence signalling in relation to partial resistance to p. hordei in barley.  transcriptomic signatures of response to p. hordei plant defence responses involve transcriptional activation of a plethora of specific genes and regulation of their temporal and spatial expression. to investigate the genome-wide transcriptional signatures of susceptible and partially resistant barley lines l and vada respectively in response to p. hordei infection, we compared ph-infected with mock-inoculated leaf material. a stringent threshold with fold change > and false discovery rate < was adopted for declaring significant differences. at this threshold, and genes were respectively identified in l and vada as'significantly differentially expressed' with overlapping between the two lines while were present in only one of the two parents. this yielded a total of genes which we considered'ph-responsive'. close examination of the expression data of the'ph-responsive' genes from both parents showed that while a substantial number failed to meet the stringent thresholds applied they still exhibited statistically significant differential expression in both parents. therefore, a relaxed threshold ignoring the fold changes was adopted for the follow-up analysis on the commonality and specificity of response to ph-infection between the resistant and susceptible lines using all ph-responsive genes. we plotted the log-transformed expression ratios of vada against l and classified them into four groups. genes that showed the same expression patterns and expression changes at p < in both lines were defined as being common to both lines, whereas, those that showed significant expression changes in one line but no significant expression changes or a contrasting expression pattern in the other line were considered as being line-specific. the remaining genes that had no strong evidence to suggest either commonality or specificity were grouped into'not determined'. there were a total of and genes that appeared to be in common, vada- or l94-specific or'not determined' representing 73%, 3%, 4% and 20% of the ph-responsive genes respectively. figure shows a colour-coded heat map that was converted from the relative expression ratios of the genes and the line-specific genes showing the overall similarity and specificity of gene expression in l and vada. full expression information of the genes and the line-specific genes is given in the additional file and respectively. genome-wide ph-responsive genes have previously been investigated using steptoe and morex, two barley cultivars with similar, intermediate levels of resistance to p. hordei. we therefore compared the data from l94/vada with those from st/mx. at exactly the same thresholds a total of genes were identified as ph-responsive in st/mx. applying exactly the same criteria as described above, we identified,, and genes that were common, st-specific and mx-specific respectively. we then explored the common genes in each of these categories between the two experiments. % of the genes detected with l94/vada were also detected with st/mx and more than half of the genes were significant in all four lines, highlighting the similarity of response to ph-infection across genotypes. of the and genes that were specifically detected in vada and l respectively, and six of these were reproducibly identified as ph-responsive in steptoe or morex. all of the vada -specific genes were up-regulated in vada and st or mx. ten of these genes showed significant differential expression between st and mx. of the six l -specific genes, only one up-regulated gene showed significant differential expression between st and mx. note: ph-responsive genes were selected on criteria with fold change > and fdr <; genes with similar patterns were selected from ph-responsive gene on p < without considering fold changes. to further characterise the biological processes represented by the ph-responsive genes, we performed gene ontology analysis by classifying the ph-responsive genes into functional biological categories based on go terms retrieved from their rice homologues through the rice database at http://rice.plantbiology.msu.edu/annotation_pseudo_goslim.shtml. the ph-responsive genes were associated with a broad range of biological processes. the primary category was related to defence response. we further classified these genes into major functional categories following the go terms in'biological process' with all remaining genes grouped into'other functions' or'unknown'. the results are shown in figure they indicate that at the sampling time point of hpi, the plants had responded to defend against the ph-infection. differential expression analysis between ph-infected recurrent parents we performed genome-wide differential expression analysis by comparing expression differences between ph-infected l and ph-infected vada at hpi. a total of genes were identified as being differentially expressed, of which were ph-responsive genes as described above. the majority represent genome-wide, genotype-specific differences in gene expression. the detailed information of these genes regarding their expression ratios, p-values and functional annotations is presented in additional file. differential expression between ph-infected qtl-nils and ph-infected recurrent parents to identify qtl-specific and differentially expressed genes accounted for by genetic differences in the qtl regions, the two reciprocal qtl-nils were compared with their respective recurrent parents: l vs. l94-rphq2+ and vada vs. vada-rphq2+ a total of genes were identified as significant in at least one comparison. of these, genes showed a significant difference in one recurrent parent/qtl-nil comparison but not with the other. we attribute these observations to the different size and incomplete overlap of the introgressed segments in the two recurrent parent/qtl-nil pairs. these genes were, therefore, not pursued further. the remaining genes showed expression differences at p < in both comparisons and were, therefore, considered potentially relevant to the qtl regions. this suggests that differential expression results from genetic factors differing specifically within the qtl regions. of these genes, were present on the list of differentially expressed genes between ph-infected vada and ph-infected l the remaining five genes did not fulfil the criteria set for the differential expression between the two ph-infected recurrent parents, but their expression differences were still statistically significant. transcription of qtl-specific and differentially expressed genes in response to ph-infection to identify whether the qtl-specific and differentially expressed genes were also ph-responsive, expression data from the ph-infected vs. mock-inoculated experiment was re-investigated. six genes showed changes that fulfilled the criteria set for defining ph-responsive genes. twelve genes did not fully meet the criteria, but their level of differential expression was still statistically significant in at least one of the lines. the others were not statistically significant. identification of positional candidates for rphq and rphq3 to determine the map position of the qtl-specific and differentially expressed genes, we took advantage of available datasets previously generated in three different eqtl studies and ph-infected seedling leaves. of the genes had one or more eqtl in at least one of these three experiments, yielding eqtl in total. the distribution of these eqtl was investigated by plotting their map positions against their lod/lrs values. genes with eqtl mapped to within the qtl regions, of which had lod > or lrs > suggesting they are cis-eqtl. we then explored three available gene-based mapping datasets: illumina opa-snps, single feature polymorphisms and tdms to help assign genetic map positions to the genes. this allowed four and nine genes to be placed within the confidence intervals of rphq and rphq respectively. all of these genes overlapped with the eqtl except two for which no eqtl was detected in the three eqtl studies. rphq and rphq on chromosome 2h and 6h are syntenic to regions on rice chromosomes os and os respectively. conservation of synteny allowed us to infer the approximate map positions of an additional genes to within the qtl regions. thus, of the qtl-specific differentially expressed genes, the map location of nine and fell within rphq and rphq respectively, whilst the others remain unknown. of note was the observation that one gene, encoding an ethylene-responsive transcription factor, has been mapped as illumina opa-snp marker 11_ to chromosome 1h at position cm. this map position is consistent with a location based on conservation of synteny between rice os and barley chromosome 1h, suggesting that differential expression of this gene is the consequence of trans-regulation by a gene located within either rphq or rphq discussion in this study, we performed differential expression analysis of two reciprocal qtl-nils and compared them with their respective recurrent parents. as qtl-nils differ genetically from their recurrent parent only in the selected qtl regions, we would anticipate that genetic polymorphism between these qtl regions would account for any differential expression observed. however, due to the complexity of gene regulation, differentially expressed genes may not necessarily be located in the introgressed qtl regions, which may themselves contain regulatory genes affecting the expression of other genes spread throughout the genome. we therefore established the map positions of differentially expressed genes by exploiting previously generated gene mapping datasets. of the genes highlighted in our comparisons between nils and recurrent parents, detected eqtl in at least one of the three previous eqtl studies and co-located at the qtl regions, most having high lod/lrs scores. eqtl with high lod scores have been demonstrated previously to be almost always cis-eqtl placing these genes within the rphq or rphq qtl regions. the observation that so many significantly differentially expressed genes appeared to be regulated in cis- is in agreement with previous studies. an exception was unigene encoding hverf this gene mapped to cm on chromosome 1h, consistent with the position of its rice homologue os05g predicted by conservation of synteny. this observation raises the possibility that the introgressed regions at either rphq or rphq contain a polymorphic trans-acting regulator that differentially modulates expression of hverf no eqtl for hverf was detected at the regions corresponding to rphq or rphq in the st/mx dh mapping population, consistent with the fact that it does not segregate for rphq or rphq hverf is a member of a family of plant transcription factors functionally involved in defence signalling pathways related to ethylene, jasmonic acid and abscisic acid. over-expression of arabidopsis aterf represses the expression of pathogenesis-related genes such as basic chitinase and beta- -glucanase genes and genes containing a gcc-box, the core sequence element of promoters required for responsiveness to ethylene. in our previous experiment with steptoe and morex, cultivars with similar but intermediate levels of partial resistance to leaf rust, we also observed that hverf was significantly up-regulated by ph-infection but no differential expression was observed between the parents. here, hverf was induced in ph-infected l and vada as compared to mock-inoculated controls, and the expression level of the vada allele was only a third of that of the l allele after induction. the association of resistance/susceptibility with lower/higher expression of hverf appears to be in agreement with the negative regulatory role of hverf on the expression of pr and other defence responsive genes. however, consistent association of higher expression of pr genes with resistance was not observed in vada and l this may reflect the general complexity of natural resistance response coupled with allelic variation at pr genes between these two lines. while this train of inference highlights hverf as potentially important in this specific defense interaction, none of the so far reported qtl for partial resistance to leaf rust, nor any of the qtl for resistance to heterologous rusts is co-located with hverf at cm on chromosome 1h. thus, hverf is not a positional candidate for any of the reported qtl. however, of direct relevance is a previously highlighted eqtl hotspot for genes that was associated with opa-snp 11_ at cm on chromosome 1h spanning the region containing hverf this hotspot comprised eqtl in less than a cm interval and contained genes primarily involved in defence response. given its known role in pr-protein regulation, we speculate that hverf represents a key regulatory relay component of the signalling pathway that controls expression of at least a portion of the genes with eqtl located at the hotspot on chromosome 1h. considering these observations together we hypothesise that the causal genetic polymorphism at either rphq or rphq differentially regulates hverf in trans, the consequence of which is differential regulation of down-stream defence responses. in this scenario, the candidate genes for rphq or rphq would be those acting up-stream rather than down-stream of hverf and possibly involved directly or indirectly in plant hormone signalling pathways. while we did not find such a candidate from the annotated functions of the qtl-specific and differentially expressed candidates for rphq and rphq the gene controlling expression of hverf may, however, not be differentially expressed between l and vada, may not be on our expression platform or may not be at the orthologous position in rice. an alternative to identifying the causal gene for rphq or rphq could be through map-based cloning of the trans-eqtl for hverf marcel et al. narrowed down the genetic interval for rphq to cm corresponding to a physical length of kb in barley and a kb syntenic region on rice chromosome inspection of all predicted genes in the rphq syntenic interval in rice identified a cluster of six peroxidase genes and a map3k gene as potential candidates because of their functional involvement in defence responses. in this study, we identified four barley genes at rphq that were differentially expressed and had homologues located in the syntenic region in rice, unigene, unigene and unigene ). unigene and both encode peroxidases and are within the cm interval containing rphq the other two, according to the fine mapping data of marcel et al. fell just outside the candidate interval. however, given the frequent breakdown in conservation of synteny, positional candidate gene identification using this approach alone remains problematic. differential expression in the qtl-nils identified an additional five candidate genes that were not apparently present in the syntenic region of rice. two of these encode proteins that are functionally involved in signal transduction, one pr protein, and one with homology to human nipa implicated in prader-willi/angelman syndrome. one gene showed no homology to known genes. thus, these five genes, together with the two peroxidase genes, are potential positional candidates for rphq further refinement of the candidate gene list will require knowledge of the role of these genes in defence response and correlation of transcript levels with resistance/susceptibility. many more genes were identified as being differentially expressed and located at the rphq region. this is expected given the larger interval of the qtl and rphq may, therefore, account for differential expression of most of the genes with unassigned map positions. functionally, none of the differentially expressed genes at rphq or rphq appear to be obvious candidates for a regulator of hverf many defence genes encoding pr proteins and components of the phenylpropanoid pathway such as phenylalanine ammonia lyase were, as would be expected, ph-responsive. pr genes encoding beta- -glucanases, chitinases and thaumatin-like proteins exhibit strong in vitro anti-fungal activity and numerous studies have shown that transgenic plants expressing pr-proteins have significant improvement of disease resistance. pal, the first committed enzyme in the phenylpropanoid pathway, is involved in synthesis of both phytoalexins and lignin. phytoalexins are antimicrobial while lignin synthesis contributes to formation of papillae, which are physical barriers against cell wall penetration by the pathogen. as part of the general response to pathogen infection, few of the genes fell into these categories co-located at the two qtl for partial resistance. one exception, unigene encoding a chitinase, co-located at rphq was ph-responsive and differentially expressed between rphq and rphq however, it was not prioritized as a candidate for rphq since the higher level of gene expression was associated with the susceptibility allele rphq whereas a number of defence genes were activated in response to ph-infection, none was found to be a promising candidate for rphq or rphq our results support the notion that components of the general defence response have incremental, rather than deterministic, roles in the outcome of an interaction between a plant and a pathogen. many attempts to identify genes for disease resistance have highlighted those involved in signal transduction or physiological and cellular functions rather than defence per se. ph-infection triggers a broad range of biological responses with defence response genes being significantly over-represented. of note is a set of genes encoding receptor-like kinase, receptor-like proteins, wrky, mapk and pr proteins, which form a complete and well-explored defence signalling cascade starting with the perception of pamps, activation of wrky transcription factors and the subsequent induction of pr proteins. our results also suggest that, in the absence of cognate r genes to p. hordei, plants still mount reactions similar to r-gene mediated responses as indicated by the significant up-regulation of genes coding for r gene proteins and marker genes for oxidative burst such glutathione s-transferase and peroxidase. although no obvious r genes were identified as candidates for the qtl in this study, r gene-like mediated responses may contribute to basal resistance as a complementary mechanism to pamp-triggered defence responses. support for this is provided by observations that resistance qtl are often coincident with the location of r-gene homologues and that mutated r genes can induce a resistance phenotype similar to quantitative resistance controlled by multiple genes. one striking characteristic of the responses to ph-infection was the activation of signalling pathways related to a broad range of plant hormones including ethylene, gibberellins, auxin, and brassinosteroid as indicated by the up-regulation of genes encoding ethylene-responsive transcription factors, acc oxidase, auxin-responsive proteins, brassinosteroid insensitive 1-associated receptor kinase, gibberellin receptors and a della protein. all of these hormones have been reported to be involved in plant defence responses and various defence pathways are interconnected through hormone-mediated signalling pathways forming complex regulatory networks. here, the identification of the ethylene-responsive factor hverf as a putative link between pathogen perception and response is consistent with a role for differential hormone signalling in partial resistance. understanding the role of rphq of rphq in initiating and coordinating the response requires further work. substantial overlap of ph-responsive genes was identified in super-susceptible and partially-resistant lines. over 70% of ph-responsive genes were detected in both l and vada and had the same expression patterns in both lines. an even higher percentage of overlapping ph-responsive genes was discovered in both steptoe and morex, two cultivars with similar and intermediate level of partial resistance. given that these lines are genetically diverse, we conclude that barley lines without known cognate r genes to p. hordei exhibit similar responses at the transcriptional level, and that observed differences are largely quantitative. similar findings have been observed in the comparison between compatible and incompatible interactions. a small proportion of ph-responsive genes in this study did appear to be resistant/susceptible line-specific and it may be that they determine part of the observed phenotypic differences between lines. however, in ph-infected leaves we found no evidence for their differential expression in the comparisons between the two qtl-nils and their respective recurrent parents. therefore, if the variation in resistance, accounted for by rphq or rphq is regulated at the transcriptional level, these are not strong candidate genes. we generated a robust expression data set in reciprocal rphq2/rphq qtl-nils at hpi, which is the timepoint previously described as being the most critical during p. hordei invasion in barley. however, we realise that transcriptional re-programming in response to pathogen infection is a dynamic and complex process and that defence-associated genes respond to input stimuli with different timing and amplitude. a limitation of our experiment is, therefore, that defence response scenarios constructed on the transcriptional profiles of the ph-responsive genes identified here is simply a snapshot of a dynamic process, at the point when infection hyphae have just attempted penetration of the host cells forming haustoria. to extend our understanding of the complex regulatory mechanisms occurring during defence against p. hordei, a more comprehensive investigation would involve sampling at multiple timepoints covering the whole infection period.  differential expression with qtl-nils identifies genes predominantly located at the target region providing both transcriptional and positional candidate genes underlying the qtl. positional analysis of the differentially expressed genes relative to the qtl has the potential to discover regulatory relays initiated from genes within the qtl. 
one gene, encoding the transcriptional repressor ethylene-responsive element binding factor was located outside the qtl at cm on chromosome 1h, within a previously detected eqtl hotspot for defence response. the results indicate that rphq or rphq contains a trans-eqtl that modulates expression of hverf we speculate that hverf functions as an intermediate that conveys the response signal from a gene contained within rphq or rphq to a host of down-stream defense responsive genes. our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of ph-responsive genes and that hormone-related signalling pathways are actively involved in response to puccinia hordei. the barley-puccinia hordei pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci for partial resistance. the molecular mechanism underpinning partial resistance in these qtl-nils is unknown. an agilent custom microarray consisting of probes derived from barley consensus est sequences was used to investigate genome-wide and qtl-specific differential expression of genes hours post-inoculation with puccinia hordei. differential gene expression between qtl-nils identifies genes predominantly located within the target region providing both transcriptional and positional candidate genes for the qtl. genetically mapping the differentially expressed genes relative to the qtl has the potential to discover trans-eqtl mediated regulatory relays initiated from genes within the qtl regions. a total of genes were identified as being significantly differentially expressed across the genome, of which were accounted for by the genetic differences defined by qtl-nils at rphq and rphq these genes were predominantly located at the qtl regions and are, therefore, positional candidates.
 the barley-puccinia hordei pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci for partial resistance. reciprocal qtl-specific near-isogenic lines have been developed that combine two qtl, rphq and rphq the largest effects detected in a recombinant-inbred-line population derived from a cross between the super-susceptible line l and partially-resistant line vada. the molecular mechanism underpinning partial resistance in these qtl-nils is unknown.  an agilent custom microarray consisting of probes derived from barley consensus est sequences was used to investigate genome-wide and qtl-specific differential expression of genes hours post-inoculation with puccinia hordei. a total of genes were identified as being significantly differentially expressed across the genome, of which were accounted for by the genetic differences defined by qtl-nils at rphq and rphq these genes were predominantly located at the qtl regions and are, therefore, positional candidates. one gene, encoding the transcriptional repressor ethylene-responsive element binding factor was located outside the qtl at cm on chromosome 1h, within a previously detected eqtl hotspot for defence response. the results indicate that rphq or rphq contains a trans-eqtl that modulates expression of hverf we speculate that hverf functions as an intermediate that conveys the response signal from a gene contained within rphq or rphq to a host of down-stream defense responsive genes. our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of ph-responsive genes and that hormone-related signalling pathways are actively involved in response to puccinia hordei.  differential gene expression between qtl-nils identifies genes predominantly located within the target region providing both transcriptional and positional candidate genes for the qtl. genetically mapping the differentially expressed genes relative to the qtl has the potential to discover trans-eqtl mediated regulatory relays initiated from genes within the qtl regions. 