BACKGROUND
clubroot, caused by the biotrophic protist plasmodiophora brassicae woronin, is one of the most serious diseases of brassica crops worldwide  <cit> .
in western canada, clubroot disease has become a major threat to the production of canola   <cit> , where more than 8 m ha of canola crops are grown annually  <cit> .
the pathogen is able to survive for up to 20 years in soil  <cit>  and many conventional disease-management measures, including cultural techniques and application of fungicides, are not effective  <cit> .
genetic resistance is the most effective and economical approach to clubroot management on canola.
european fodder turnips  are the major source of clubroot-resistance  genes, which have been introduced into other brassica crops including oilseed rape , rutabaga  and chinese cabbage  .
since  <dig>  several resistant  canola cultivars have been released in canada, and all of them carry a single dominant cr gene.
the source and genetic information are not revealed for these cr genes  <cit> .
the durability of these clubroot r cultivars remains unknown in western canada, but resistance conferred by a single gene is generally not durable.
breakdown of clubroot resistance has been reported on chinese cabbage  <cit>  and oilseed rape  <cit> .
a resistant canola cultivar showed substantially increased clubroot severity after being exposed to pathotype  <dig> of p. brassicae after only two cycles under controlled conditions  <cit> .
rotation or pyramiding of cr genes with different mechanisms of resistance may be used to increase the durability of clubroot resistance if a diverse group of cr genes can be identified and their resistance mechanisms characterized.
our prior work evaluated  <dig> brassica accessions and identified a range of cr candidates from b. rapa, b. nigra and b. oleracea <cit> .
most of the known cr genes have been identified from b. rapa, with eight loci reported previously: crr <dig>  crr <dig>  crr <dig>  crr <dig>  cra, crb, crc and crk.
cra and crr <dig> have been isolated recently  <cit> .
another cr gene, rpb <dig>  was identified from arabidopsis thaliana ecotype tsu- <dig>  <cit> , but there has been no further report on its orthlogs in other arabidopsis ecotypes.
a new cr gene  was identified recently from the cv.
flower nabana  of pak choy  via rough mapping  <cit> .
rpb <dig> is identical to rcr1described in this paper, and the name change was to avoid potential confusion with the rpb <dig> from arabidopsis.
there has been little information on molecular mechanisms associated with any of the cr genes reported.
in a. thaliana, host metabolism was altered by p. brassicae infection; transcriptome studies based on microarray analysis showed that genes encoding enzymes involved in carbohydrate metabolism were upregulated in root tissues of the susceptible  col- <dig> ecotype  <cit> , but not in moderately resistant  ecotypes which appeared to reduce or delay pathogen-triggered metabolic diversion and cell enlargement or proliferation in the host  <cit> .
reduced trehalose and arginine metabolism were also reported with the partially resistant a. thaliana ecotype bur- <dig> when compared with that in a susceptible ecotype  <cit> .
secondary metabolism, including flavonoids, may also contribute to formation of characteristic club symptoms in arabidopsis, and inhibition of oxoglutaric acid-dependent dioxygenases reduced club development  <cit> .
treatment with the phytohormone salicylic acid or biofungicides reduced clubroot development on a. thaliana and b. napus via activation of several defense-related pathways in the hosts .
however, there is no information on molecular mechanisms of clubroot resistance in brassica species based on transcriptome analysis.
rna sequencing  has been employed recently to elucidate resistance mechanisms involved in plant-pathogen interactions including sclerotinia homoeocarpea-creeping bentgrass  <cit>  and phytophthora infestans-potato tuber  <cit> .
in the present study, we intended to: 1) identify and characterize the cr gene from a highly resistant pak choy cultivar using genetic mapping; 2) develop molecular markers closely linked to this cr gene to facilitate marker-assisted selection  at the young seedling stage; and 3) analyze the global transcriptome profile associated with the cr gene based on rna-seq.
we examined differential gene expression between r and s f <dig> plants, and the result provided important insights into the molecular mechanisms of clubroot resistance.
this work also sets the first step toward the development of canola germplasm using cr genes with potentially different modes of action against clubroot.
RESULTS
the clubroot resistance in cv.
fn is associated with a single dominant allele
all of the fn plants were resistant to pathotype  <dig> of p. brassicae, showing no clubroot symptom at 5 weeks after inoculation, whereas all of the acdc plants were susceptible .
analyses of the f <dig> populations from reciprocal crosses showed a segregation pattern that would fit a 1: <dig> ratio between r and s plants , indicating that the resistance in fn is associated with a single dominant nuclear gene.
this gene was designated as rcr <dig> .
also, the pattern of clubroot disease response in parental and f <dig> populations  indicated that the rcr <dig> locus was likely heterozygous in the cv.
fn.figure  <dig>
segregation in clubroot resistance for parents  and f
1
populations derived from reciprocal crosses .
these f <dig> plants were not part of the f <dig> population  used later for fine mapping of cr genes.
fine mapping of the gene rcr1and development of molecular markers
rcr <dig> was roughly mapped to a range of  <dig>  cm in the b. rapa linkage group a <dig> flanked by the markers sn <dig> and sr6340i , and fine mapping was based on testing additional  <dig>  f <dig> plants using pathotype  <dig> of p. brassicae and on analysis using these flanking markers .
the flanked segment is homologous to the region between  <dig>  mb and  <dig>  mb on the a <dig> , with  <dig> genes annotated  and five of them  identified as encoding toll interleukin- <dig> receptor - nucleotide-binding site -leucine-rich repeat  class of proteins .
bra <dig> is located close to  <dig>  mb and the rest were in a cluster located between  <dig>  mb and  <dig>  mb.figure  <dig>
linkage maps of the regions in which the
rcr1
gene is located.
broken lines drawn regions defined by different molecular markers on
b. rapa
linkage group a <dig>  a) rough mapping of rcr <dig> based on a small f <dig> population  derived from acdc × fn.
the genetic distance is shown on the left.
b) fine mapping of rcr <dig> based on  <dig>  f <dig> plants.
c) physical locations in mb  of the molecular markers and tir-nbs-lrr genes in the region flanked by the markers sn <dig> and sr6340i.
a total of  <dig> recombinants were identified via comparison of marker and phenotype data over the  <dig>  f <dig> plants , with  <dig> falling between sn <dig> and rcr <dig> and  <dig> between rcr1and sr6340i.
a caps marker , homologous to bra <dig> at  <dig>  mb, was developed for further analysis of the  <dig> recombinants, and showed an approximate distance of  <dig>  cm from rcr <dig>  which was closer to the cr gene than sr6340i.
the interval flanked by sn <dig> and a3- <dig> was estimated at  <dig>  cm, consisting of approximately  <dig> kb with  <dig> genes annotated .
the caps marker ms7- <dig>  was identified slightly later and appeared even closer to rcr <dig> than a3- <dig> .
based on the rate of recombination, the genetic distance of rcr <dig> was estimated at  <dig>  cm from sn <dig> and  <dig>  cm from ms7- <dig>  respectively.
the cluster of four tir-nbs-lrr genes and one defense-related gene  are located also within this interval.
the gene ontology  terms for these genes are in table  <dig> and figure  <dig> figure  <dig>
genotypes and phenotypes of recombinants selected from the mapping population inoculated with pathotype  <dig> of
plasmodiophora brassicae
.
line identifications and phenotypes  are denoted on the left and right, respectively, with marker names at the top.
resistance alleles are denoted in light grey and susceptible alleles in black.
the two markers in a grey shadow flank the narrowest interval containing the rcr <dig> gene.
the defense-related genes annotated within the fine mapped region flanked by the markers sn <dig> and a3- <dig> in the
brassicae rapa
linkage group a <dig> and their associated gene ontology  terms
bra019401
bra019407
bra019409
bra019410
bra019412
bra019413
bra038776gene ontology  annotations of genes residing in the region flanked by the markers sn <dig> and sr6340i in fine mapping: go terms in the category of a) biological process and b) molecular functions.
the value labeled in the pie chart of both a) and b) are the number of genes annotated with the corresponding go term.
validation of selected markers for detection of rcr1in backcross populations
on the bc1b.
napus population, sn <dig> detected rcr <dig> in  <dig> % of the resistant  and  <dig> % of susceptible  plants, while sr6340i detected the cr gene in  <dig> % of r and  <dig> % of s plants, respectively .
on b. rapa, however, the accuracy was slightly poorer for both sn <dig>  and sr6340i .
the accuracy was much poorer for the markers sb4889b and ss <dig> on both b. napus and b. rapa, with erroneous identification of rcr <dig> at > <dig> %.table  <dig>
validation of flanking markers for detecting the
rcr1
gene  in clubroot resistant bc
1
progeny
b. napus
a
b. rapa
a
b
b
athe bc <dig> populations were derived from crosses of a dh line of b. napus  and b. rapa , respectively, with cv.
fn.
each bc <dig> population use for the experiment consisted of  <dig> plants.
b“resistance” and “susceptible” are phenotypical reactions to pathotype  <dig> of p. brassicae.
the percentage indicates the rate of rcr <dig> identification in plants using the marker.
transcriptome profiling based on rna-seq
inoculated f <dig> seedlings from the cross acdc × fn were bulked  based on mas and examined for global transcriptomes using rna-seq.
approximately  <dig> million raw reads were generated from a total of six pooled samples.
about 92% of them passed the quality control standard, yielding  <dig> million of clean reads .
about 60% of the total reads were mapped to the b. rapa reference genome, with 97% of them being uniquely mapped, while 33% of the total reads were unmapped.table  <dig>
summary of the rna-sequencing reads from inoculated resistant and susceptible
b. rapa
root samples
a total of  <dig>  genes were annotated in the b. rapa reference genome sequence .
transcripts of  <dig>  of these genes were detected based on rpkm calculations , and among them, more than 75%  had a coverage of 90% or higher by the mapped reads.
a total of  <dig>  differentially expressed genes  were identified in this study , with  <dig>  genes upregulated and  <dig> down-regulated in the r samples relative to s samples.
almost all genes in the fine mapped rcr <dig> region between  <dig>  mb and  <dig>  mb of a <dig> were expressed, but only a few of them were identified as degs and most of them showed no difference in expression levels between inoculated r and s. interestingly, two of the tir-nbs-lrr genes  within this region were significantly upregulated in the inoculated r treatment relative to the inoculated s treatment.
rt-qpcr analysis of  <dig> selected genes over the same r and s bulk samples showed a trend consistent with that of rpkm calculations and statistical analyses of transcript data .
the rt-qpcr data confirmed up-regulation of a class- <dig> non-symbiotic hemoglobin , erd <dig> protein , protein tify 10a , s-adenosyl-l-methionine:carboxyl methyltransferase family protein , transcriptional factor bhlb92-like protein  and transcriptional factor bhlh <dig>  genes in resistant samples detected via rna-seq.
the data also verified down-regulation of a chitinase-like protein , ralf-like  <dig> protein , endochitinase isolog  and cell-wall-protein-like protein  genes  as indicated in rna-seq analysis.figure  <dig>
validation of rpkm-calculated expression ratios for selected differentially expressed genes  using rt-qpcr.
rpkm values from rna-seq are denoted in black, and rt-qpcr results in while.
capped lines represent the standard deviations from three biological replicates.
annotation of degs
the degs were functionally annotated based on go terms  and sorted into the go-term biological process, molecular function and cellular component  using blast2go  <cit> .
the statistics for go-term mapping were provided in additional file 1: figure s <dig>  a total of  <dig> degs retrieved no hits with blast .
the annotated degs with upregulated patterns in r samples fell mainly into  <dig> categories of biological process .
the go term “defense response”  was also one of the major categories identified.
other upregulated biological processes of go terms included signal transduction, various metabolic/biosynthetic processes and regulation of metabolic processes.
for molecular functions, several cellular-component go terms were identified, especially those associated with the plasma membrane representing the largest group .
the go terms for biological processes associated with down-regulated degs were mostly in the category of “anatomical-structural and multicellular-organismal development”  and “regulation of primary metabolic process”.
most of the molecular functional go terms associated with down-regulated degs were in the same categories as those of upregulated degs, although several unique terms were identified, including sequence-specific dna binding transcription factor activity, hydrolase activity on o-glycosyl compounds, substrate-specific trans-membrane transporter activity, and nucleoside-triphosphatase activity .
similarly, the majority of cellular-component go terms of down-regulated degs fell into categories similar to those of upregulated degs, with only three new go terms observed: vacuole, chloroplast stromal and organelle membrane .figure  <dig>
go terms associated with upregulated degs.
a) go terms in the biological process category.
b) go terms in the molecular functions.
c) go terms in cellular components.
the values labeled in the pie charts of panel a, b and c are the percentage of degs annotated with the corresponding go term relative to the total degs.
go terms associated with down-regulated degs.
a) go terms in the biological process category.
b) go terms in molecular functions.
c) go terms in cellular component.
the values labeled in the pie charts of panel a, b and c are the percentage of degs annotated with the corresponding go term relative to the total degs.
transcription factors  were also characterized broadly for degs; a total of  <dig> upregulated and  <dig> down-regulated degs were grouped into seven types of tf, based on their conserved structures.
for upregulated degs,  <dig> of them belong to wrky,  <dig> are myb domain-containing tfs,  <dig> are ethylene -responsive tfs,  <dig> are bhlh-domain containing tfs,  <dig> belong to the ap2/erf family,  <dig> are heat-stress related tfs, and the remaining  <dig> belong to “other” tf families .
for down-regulated degs,  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> of them fell into the respective tf families .table  <dig>
up-regulated differentially expressed genes  annotated as transcription factors
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra013584
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra001202
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra017656
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra030208
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra011002
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra012828
bra <dig>  bra <dig>  bra <dig> bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra015582
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra030178
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra038774
bra <dig>  bra002168
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra008716
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra008460
bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra <dig>  bra014971
biological-process go terms for up- and down-regulated degs
analysis using the fisher’s exact test in blast2go identified the enrichment associated with up- and down-regulated degs; a total of  <dig> biological-process go terms displayed significant enrichment , with  <dig> of them associated with upregulated and  <dig> with down-regulated degs.
most of these enriched go terms were related to “responses”, including those to chemical and hormone stimuli.
the results were similar for metabolic-process go terms, with most of the enriched term being related to “responses”.
among  <dig> enriched go terms for upregulated degs,  <dig> were related to lipid metabolism, including lipid metabolic process, cellular lipid metabolism, lipid biosynthesis, fatty acid metabolism, fatty acid biosynthesis, oxylipin metabolism and biosynthesis.
several go terms related to defense-related phytohormones, including jasmonic acid and ethylene, but not salicylic acid , were also highly enriched in inoculated r plants relative to those in inoculated s plants .
a total of  <dig> degs were annotated under go terms with sa-related biological processes, including “response to sa stimulus”, “sa biosynthetic process “systemic acquired resistance, sa-mediated signaling pathway” etc.
, but none of them was significantly enriched in either up- or down-regulated go terms, as determined with the fisher’s exact test.
noticeably, genes associated with several common defense responses were significantly upregulated, including “callose deposition”, “defense response by callose deposition” and “indole-containing compound metabolic process”.
in contrast, most of the biological-process go terms enriched in down-regulated degs were related to “development” and “morphogenesis”, including developmental process, anatomical structure development, anatomical structure morphogenesis and uni-dimensional cell growth .figure  <dig>
comparison of go annotations  of up- and down-regulated degs.
blue bars represent the upregulated degs while red bars represent down-regulated degs.
the values were the percentage of degs annotated with the corresponding go terms relative to the total up- or down-regulated degs.
statistics of enrichment analysis are presented in the additional file 1: table s <dig>
transcript analysis of selected defence-related degs using rt-qpcr
based on rna-seq analysis,  <dig> strongly upregulated and  <dig> strongly down-regulated degs , involved possibly in resistance based on the functional annotation , were subjected to rt-qpcr analysis.
all of the  <dig> upregulated degs displayed similar transcriptional patterns; the transcript levels, based on their relative quantity, were comparable for r and s plants without pathogen inoculation but significantly higher in r plants after inoculation .
the  <dig> down-regulated degs, however, showed different transcriptional patterns; bra <dig> and bra <dig> displayed comparable transcript levels in non-inoculated s and r plants but these genes were significantly induced by p. brassicae in s plants and suppressed in r plants .
bra <dig> and bra <dig> did not show significant induction by the pathogen in s plants relative to those in non-inoculated s or r plants, but suppressed in inoculated r plants .
two of the tir-nbs-lrr genes  residing in the rcr <dig> region were also identified as degs in the rna-seq, but were not included in the rt-qpcr test due to only moderate rpkm values.
preliminary rt-qpcr trials on degs with moderate rpkm showed unsatisfactory amplification .figure  <dig>
the relative transcription quantity  measured with rt-qpcr for selected upregulated degs identified in rna-seq.
the treatments were s-susceptible  and r-resistant  plants inoculated with plasmodiophora brassicae or water .
the vertical axis represents rtq against an endogenous control .
treatments with one asterisk showed significantly higher rtq  than those without asterisk, but lower rtq than those with two asterisks.
the relative transcription quantity  measured with rt-qpcr for selected down-regulated degs identified in rna-seq.
the treatments were s-susceptible  and r-resistant  plants inoculated with plasmodiophora brassicae or water .
the vertical axis represents rtq against an endogenous control .
treatments with one asterisk had a significantly different level of rtq  relative to without asterisk or with two asterisks.
discussion
mapping of the rcr1gene and development of genetic markers
the cr gene rcr <dig>  was mapped previously to a genomic region in the linkage group a <dig> flanked by the markers sn <dig> and sr6340i  <cit> .
additional markers were developed in the current study using the b. rapa genome information, with the markers a3- <dig> and ms7- <dig> being much closer to the cr gene than sr6340i.
several markers were highly accurate in detecting rcr <dig> in both b. rapa and b. napus plants, especially when two flanking markers were used together.
these markers will be useful for mas in resistance breeding.
rcr <dig> is the only cr gene reported in pak choy , but the original source of the gene is not known.
most of the cr genes identified in chinese cabbage  originated from european turnip  <cit> .
four cr genes have been mapped previously to the linkage group a <dig>  including cra <cit> , crb <cit> , crk <cit>  and crr <dig> <cit> .
cra has also been cloned recently  <cit> .
based on the relationship between common markers and the location of these cr loci, diederichsen et al.
<cit>  suggested that cra and crr <dig> are identical, allelic or at least closely linked to crb and crk.
recent work also indicated that crb is likely in the same position as cra, and both genes conferred resistance to pathotypes  <dig> and  <dig> but not to pathotypes  <dig> and  <dig> of p. brassicae <cit> .
based on the linkage distance, rcr <dig> is close to both cra and crb.
however, the cv.
fn which carries rcr <dig> is highly resistant to both pathotypes  <dig> and  <dig>  <cit> , suggesting a different resistance spectrum from that of cra or crb.
the mechanisms for clubroot resistance are not well understood.
among the  <dig> genes annotated within the region defined by sn <dig> and a3- <dig> , four tir-nbs-lrr genes can be located .
the protein family containing nbs and lrr domains is the largest class of r genes cloned so far  <cit> .
cra and crr <dig> isolated from b. rapa also encode tir-nbs-lrr proteins  <cit> .
therefore, it is possible that rcr <dig> is one or a cluster of these tir-nbs-lrr genes in the mapped region.
transcriptome analysis and go annotation of degs
transcriptome profiling can provide insights into the mechanisms of disease resistance.
this approach has been used to characterize several molecular components associated with clubroot disease development, especially the role of cytokinins on a. thaliana <cit>  and b. juncea <cit> .
however, no comparative analysis of gene transcription between clubroot resistant and susceptible plants was available.
in our study, transcripts of  <dig>  genes were analyzed via rna-seq and  <dig>  degs were identified between inoculated r and s plants.
this number of degs is comparable to that observed in a previous transcriptome analysis between rosette and folding leaves of chinese cabbage  <cit> .
fifty degs retrieved no hit in a blast search , indicating a number of unknown genes expressed in inoculated r plants.
the go terms “defense response”  and “plasma membrane”  accounted for a substantial portion of upregulated degs.
some of these genes may be candidates for further studies of clubroot resistance, because the plasma membrane is where most plant-pathogen interaction occurs   <cit> .
additionally, a large proportion of upregulated degs were annotated with the go term biological process involved in responses to external stimuli  and many of these go terms were also significantly enriched for upregulated degs .
these results indicate that many cellular activities in inoculated r plants were significantly upregulated at  <dig> dpi when root infection and pathogen colonization were taking place .
it also appears that infection may have caused differential activation or deactivation of certain genes in the host that may affect clubroot development on resistant and/or susceptible plants.
the enrichment analysis identified go terms for biological process associated with both upregulated and down-regulated degs.
several lipid compounds were implied to play a role in the inoculated r plants.
lipids have been shown to play critical roles in detecting infection and may also have a key role in regulating gene transcription  <cit> .
during infection by p. brassicae, the r plants appeared to be able to mobilize lipid biosynthesis and metabolism, because several genes involved in oxylipin biosynthetic and metabolic processes, including bra <dig>  bra <dig> and bra <dig>  were upregulated substantially .
one group of the most intensively studied oxylipins is jasmonates, likely due to their involvement in multiple plant biological processes  <cit> .
go terms associated with “response to jasmonic acid  stimulus”, “ja metabolic process” and “ja biosynthetic process” were significantly enriched within the group of upregulated degs .
this possibly indicates that jasmonates have a role in clubroot resistance mediated by rcr <dig>  prost et al.
<cit>  employed in vitro growth inhibition assays to evaluate  <dig> natural oxylipins and found that  <dig> of them had inhibitory effects on a wide range of plant pathogens.
similarly, jasmonates appear to play a role in induced resistance to clubroot caused by biofungicides  <cit> .
oxylipins may also be used by both host and pathogen as regulatory compounds during the host-pathogen interaction   <cit> .
further work is needed to confirm the specific role of oxylipins in clubroot resistance.
potential molecular mechanisms for clubroot resistance
several clubroot-resistance mechanisms identified previously were supported in the current study.
for example, genes involved in olefin  metabolic and biosynthetic processes and in response to et stimulus, were upregulated in plants carrying rcr <dig> .
et had previously been shown to restrict club development in a. thaliana <cit>  and to have a role in induced resistance mediated by biofungicides  <cit> .
this result supports the current opinion that ja and et may act synergistically in plant defense  <cit> .
reinforcement of plant cell wall was also indicated based on the enrichment of go terms for upregulated degs associated with callose localization and deposition .
the role of callose has been well documented in resistance to penetration of plant cell wall by fungal pathogens  <cit> .
in arabidopsis, genes encoding the synthesis of β- <dig>  glucan  were suppressed in a compatible interaction between p. brassicae and susceptible ecotype col- <dig>  <cit> .
in the current study, the gene bra <dig>  encoding an expansin-like protein , showed the highest increase in transcript among all  <dig>  degs upregulated.
this may indicate a structural/composition alteration to the cell wall, which could affect secondary infection in epidermal and cortical cells.
some of the above-mentioned defense-related degs were analyzed further with rt-qpcr using inoculated and non-inoculated plants to determine transcript levels as affected by p. brassicae .
the results demonstrated that the inoculation increased the expression of these genes in both s and r plants.
inoculated r plants, however, showed significantly stronger expression of these genes relative to inoculated s plants.
this result is consistent with that of rna-seq.
in addition, genes involved in synthesis of indole-containing compounds were also upregulated in r seedlings.
the anti-microbial compounds derived from this metabolic process typically include tryptophan-derived metabolites  <cit>  and flavonoids converted from aromatic-acid phenylalanine  <cit> .
these indicate potential involvement of secondary metabolites in clubroot resistance.
although several common resistance mechanisms are identified with rna-seq, further analysis is needed to assess the relative importance of each mechanism in clubroot resistance conferred by rcr <dig>
as described above, rcr <dig> may encode a tir-nbs-lrr protein.
the best characterized defense signaling pathway related to this class of r proteins involves the biosynthesis of salicylic acid  and pathogenesis-related  proteins, accompanied typically by a hypersensitive reaction   <cit> .
additionally, sa biosynthesis is linked frequently to host resistance against biotrophic pathogens  <cit> .
in the current study, however, go terms related to sa biosynthesis, metabolism or signaling processes were not significantly up-/down-regulated based on the enrichment analysis.
this lack of substantial change in transcription of genes involved in sa biosynthesis has also been observed with induced resistance against clubroot on canola  <cit> .
previous reports have suggested hr as one of the possible mechanisms for clubroot resistance  <cit> , but only two upregulated degs, i.e., bra <dig>  and bra <dig>  were identified as pr genes based on go annotation  in the current study and there is no experimental evidence to link any of them to sa- or hr-related defense responses.
in a cytological observation, deora et al.
found no evidence of hr with clubroot resistant canola cultivars  <cit> .
it is possible that sa signaling pathways may not play a critical role in clubroot resistance.
another mechanism for disease resistance is suppression of metabolic processes in the host that are required for pathogenesis.
previous research had demonstrated that the level of auxin  increased in roots during secondary infection by p. brassicae, likely as the result of enhanced biosynthesis and conversion of host auxin precursors induced by the pathogen  <cit> .
in the current study, bra <dig>  which encodes a saur family of proteins, was among the most highly induced genes  in r plants relative to s plants.
saur proteins are closely linked to auxin biosynthesis and signaling  <cit> .
for example, saur <dig> had been found to be a negative regulator of auxin biosynthesis and transport in rice  <cit> .
with clubroot, pathogen-induced auxin metabolism had been linked to pathogenesis in arabidopsis  <cit> .
additionally, several genes encoding the auxin-responsive gh <dig> family of proteins were upregulated in r plants.
the gh <dig> family has been linked with increased basal immunity via suppressing pathogen-induced auxin accumulation in rice  <cit> .
in addition to manipulating plant auxin homeostasis, over-expression of gh <dig>  in an activation-tagged mutant of arabidopsis displayed enhanced biosynthesis of camalexin, the major phytoalexin found with pathogen infection in arabidopsis  <cit> .
since the biosynthesis of camalexin and auxin were derived from the common precursor tryptophan  <cit> , the metabolic stream may have been redirected to flow into the biosynthesis of phytoalexin from auxin in the r plants.
taken together, the results indicate that pathogen-activated auxin synthesis might have been suppressed or disrupted in r plants.
most of the enriched go terms associated with down-regulated degs were related to growth and development, including cell cycle, uni-dimensional cell growth, anatomical structure morphogenesis, and cell morphogenesis .
down-regulation of these genes may play a role in the resistance mediated by rcr <dig> because hypertrophy, the most typical symptom of clubroot  <cit> , is related positively to these physiological activities.
expression of genes involved in cell enlargement and proliferation was inhibited in an arabidopsis line partially resistant to p. brassicae, relative to the susceptible reaction  <cit> .
in the current study, two of the genes  annotated for the go term “uni-dimensional cell growth” were upregulated by the pathogen in inoculated s plants , as opposed to the same genes in inoculated r plants that changed little relative to those in non-inoculated plants.
down-regulation of these degs possibly works in conjunction with up-regulation of defense-related genes described above in resulting in a resistant outcome in plants carrying rcr <dig>  it is not clear if down-regulation of growth/development degs is directly related to the suppression of auxin-dependent metabolism via enhanced expression of bra <dig> or gh <dig> genes observed.
however, the evidence indicates that one or more processes related to auxin biosynthesis and cell growth/development are disrupted in r plants, with infection by p. brassicae.
rna-seq analysis revealed that the clubroot resistance conferred by rcr <dig> involves complex mechanisms via a variety of biological processes controlled likely by corresponding transcription factors .
as expected,  <dig> upregulated degs were identified based on their conserved structures as tfs responsible for activation of several biological processes potentially involved in disease resistance .
consistent with the roles of et and ja discussed above, et-responsive tfs including the ap2/erf-family tfs   <cit>  and bhlh-family tfs   <cit>  were upregulated to relatively high levels .
wrky-family tfs were also upregulated.
their involvement in disease resistance, including effector-triggered immunity, has been suggested previously  <cit> .
myb-family proteins may be involved in secondary metabolism including flavonoid  <cit>  and secondary cell-wall biosynthesis  <cit> .
these biological functions may contribute to clubroot resistance by generating anti-microbial metabolites and strengthening host cell walls.
several heat-stress and “other” types of tfs were also identified, but their role in resistance is not understood.
it is interesting that no heat-stress tfs were identified in the down-regulated degs, but the significance of this observation is unclear.
only a small number of defense-related degs were present in the  <dig> down-regulated degs sorted into the tf groups.
the other down-regulated degs in tf groups are involved generally in cellular growth and development.
in other words, these down-regulated tfs may represent targets that p. brassicae up-regulates during pathogenesis in s plants.
CONCLUSIONS
genetic resistance is the cornerstone for management of clubroot on canola.
in this study, we characterized the cr gene rcr <dig>  based on genetic mapping and transcript analysis, to develop markers for mas and decipher molecular mechanisms of resistance associated with rcr <dig>  rna-seq analysis identified a range of biological processes potentially involved in clubroot resistance, consisting of both up-regulated defense-related and suppressed pathogenesis-related responses.
this information is highly useful to design a breeding strategy based on modes of action of cr genes to achieve strong and durable clubroot resistance.
although sa biosynthesis is often linked to plant resistance against biotrophic pathogens, genes involved in sa biosynthetic pathways were not activated in inoculated plants carrying rcr <dig>  in contrast, genes involved in ja/et and callose biosynthesis were upregulated substantially.
the biosynthesis or signaling of ja/et has not been identified previously for resistance to clubroot.
further research is needed to confirm specific roles of these phytohormones in clubroot resistance mediated by rcr <dig>  it will also be useful to look at these phytohormones in association with other cr genes.

 clubroot, caused by the biotrophic protist plasmodiophora brassicae woronin, is one of the most serious diseases of brassica crops worldwide. in western canada, clubroot disease has become a major threat to the production of canola, where more than 8 m ha of canola crops are grown annually. the pathogen is able to survive for up to 20 years in soil and many conventional disease-management measures, including cultural techniques and application of fungicides, are not effective. genetic resistance is the most effective and economical approach to clubroot management on canola. european fodder turnips are the major source of clubroot-resistance genes, which have been introduced into other brassica crops including oilseed rape, rutabaga and chinese cabbage. since several resistant canola cultivars have been released in canada, and all of them carry a single dominant cr gene. the source and genetic information are not revealed for these cr genes. the durability of these clubroot r cultivars remains unknown in western canada, but resistance conferred by a single gene is generally not durable. breakdown of clubroot resistance has been reported on chinese cabbage and oilseed rape. a resistant canola cultivar showed substantially increased clubroot severity after being exposed to pathotype of p. brassicae after only two cycles under controlled conditions. rotation or pyramiding of cr genes with different mechanisms of resistance may be used to increase the durability of clubroot resistance if a diverse group of cr genes can be identified and their resistance mechanisms characterized. our prior work evaluated brassica accessions and identified a range of cr candidates from b. rapa, b. nigra and b. oleracea. most of the known cr genes have been identified from b. rapa, with eight loci reported previously: crr crr crr crr cra, crb, crc and crk. cra and crr have been isolated recently. another cr gene, rpb was identified from arabidopsis thaliana ecotype tsu-, but there has been no further report on its orthlogs in other arabidopsis ecotypes. a new cr gene was identified recently from the cv. flower nabana of pak choy via rough mapping. rpb is identical to rcr1described in this paper, and the name change was to avoid potential confusion with the rpb from arabidopsis. there has been little information on molecular mechanisms associated with any of the cr genes reported. in a. thaliana, host metabolism was altered by p. brassicae infection; transcriptome studies based on microarray analysis showed that genes encoding enzymes involved in carbohydrate metabolism were upregulated in root tissues of the susceptible col- ecotype, but not in moderately resistant ecotypes which appeared to reduce or delay pathogen-triggered metabolic diversion and cell enlargement or proliferation in the host. reduced trehalose and arginine metabolism were also reported with the partially resistant a. thaliana ecotype bur- when compared with that in a susceptible ecotype. secondary metabolism, including flavonoids, may also contribute to formation of characteristic club symptoms in arabidopsis, and inhibition of oxoglutaric acid-dependent dioxygenases reduced club development. treatment with the phytohormone salicylic acid or biofungicides reduced clubroot development on a. thaliana and b. napus via activation of several defense-related pathways in the hosts. however, there is no information on molecular mechanisms of clubroot resistance in brassica species based on transcriptome analysis. rna sequencing has been employed recently to elucidate resistance mechanisms involved in plant-pathogen interactions including sclerotinia homoeocarpea-creeping bentgrass and phytophthora infestans-potato tuber. in the present study, we intended to: 1) identify and characterize the cr gene from a highly resistant pak choy cultivar using genetic mapping; 2) develop molecular markers closely linked to this cr gene to facilitate marker-assisted selection at the young seedling stage; and 3) analyze the global transcriptome profile associated with the cr gene based on rna-seq. we examined differential gene expression between r and s f plants, and the result provided important insights into the molecular mechanisms of clubroot resistance. this work also sets the first step toward the development of canola germplasm using cr genes with potentially different modes of action against clubroot.  the clubroot resistance in cv. fn is associated with a single dominant allele all of the fn plants were resistant to pathotype of p. brassicae, showing no clubroot symptom at 5 weeks after inoculation, whereas all of the acdc plants were susceptible. analyses of the f populations from reciprocal crosses showed a segregation pattern that would fit a 1: ratio between r and s plants, indicating that the resistance in fn is associated with a single dominant nuclear gene. this gene was designated as rcr. also, the pattern of clubroot disease response in parental and f populations indicated that the rcr locus was likely heterozygous in the cv. fn.figure segregation in clubroot resistance for parents and f 1 populations derived from reciprocal crosses. these f plants were not part of the f population used later for fine mapping of cr genes. fine mapping of the gene rcr1and development of molecular markers rcr was roughly mapped to a range of  cm in the b. rapa linkage group a flanked by the markers sn and sr6340i, and fine mapping was based on testing additional  f plants using pathotype of p. brassicae and on analysis using these flanking markers. the flanked segment is homologous to the region between  mb and  mb on the a, with genes annotated and five of them identified as encoding toll interleukin- receptor - nucleotide-binding site -leucine-rich repeat class of proteins. bra is located close to  mb and the rest were in a cluster located between  mb and  mb.figure linkage maps of the regions in which the rcr1 gene is located. broken lines drawn regions defined by different molecular markers on b. rapa linkage group a a) rough mapping of rcr based on a small f population derived from acdc × fn. the genetic distance is shown on the left. b) fine mapping of rcr based on  f plants. c) physical locations in mb of the molecular markers and tir-nbs-lrr genes in the region flanked by the markers sn and sr6340i. a total of recombinants were identified via comparison of marker and phenotype data over the  f plants, with falling between sn and rcr and between rcr1and sr6340i. a caps marker, homologous to bra at  mb, was developed for further analysis of the recombinants, and showed an approximate distance of  cm from rcr which was closer to the cr gene than sr6340i. the interval flanked by sn and a3- was estimated at  cm, consisting of approximately kb with genes annotated. the caps marker ms7- was identified slightly later and appeared even closer to rcr than a3-. based on the rate of recombination, the genetic distance of rcr was estimated at  cm from sn and  cm from ms7- respectively. the cluster of four tir-nbs-lrr genes and one defense-related gene are located also within this interval. the gene ontology terms for these genes are in table  and figure  figure genotypes and phenotypes of recombinants selected from the mapping population inoculated with pathotype of plasmodiophora brassicae. line identifications and phenotypes are denoted on the left and right, respectively, with marker names at the top. resistance alleles are denoted in light grey and susceptible alleles in black. the two markers in a grey shadow flank the narrowest interval containing the rcr gene. the defense-related genes annotated within the fine mapped region flanked by the markers sn and a3- in the brassicae rapa linkage group a and their associated gene ontology terms bra019401 bra019407 bra019409 bra019410 bra019412 bra019413 bra038776gene ontology annotations of genes residing in the region flanked by the markers sn and sr6340i in fine mapping: go terms in the category of a) biological process and b) molecular functions. the value labeled in the pie chart of both a) and b) are the number of genes annotated with the corresponding go term. validation of selected markers for detection of rcr1in backcross populations on the bc1b. napus population, sn detected rcr in % of the resistant and % of susceptible plants, while sr6340i detected the cr gene in % of r and % of s plants, respectively. on b. rapa, however, the accuracy was slightly poorer for both sn and sr6340i. the accuracy was much poorer for the markers sb4889b and ss on both b. napus and b. rapa, with erroneous identification of rcr at > %.table validation of flanking markers for detecting the rcr1 gene in clubroot resistant bc 1 progeny b. napus a b. rapa a b b athe bc populations were derived from crosses of a dh line of b. napus and b. rapa, respectively, with cv. fn. each bc population use for the experiment consisted of plants. b“resistance” and “susceptible” are phenotypical reactions to pathotype of p. brassicae. the percentage indicates the rate of rcr identification in plants using the marker. transcriptome profiling based on rna-seq inoculated f seedlings from the cross acdc × fn were bulked based on mas and examined for global transcriptomes using rna-seq. approximately million raw reads were generated from a total of six pooled samples. about 92% of them passed the quality control standard, yielding million of clean reads. about 60% of the total reads were mapped to the b. rapa reference genome, with 97% of them being uniquely mapped, while 33% of the total reads were unmapped.table summary of the rna-sequencing reads from inoculated resistant and susceptible b. rapa root samples a total of genes were annotated in the b. rapa reference genome sequence. transcripts of of these genes were detected based on rpkm calculations, and among them, more than 75% had a coverage of 90% or higher by the mapped reads. a total of differentially expressed genes were identified in this study, with genes upregulated and down-regulated in the r samples relative to s samples. almost all genes in the fine mapped rcr region between  mb and  mb of a were expressed, but only a few of them were identified as degs and most of them showed no difference in expression levels between inoculated r and s. interestingly, two of the tir-nbs-lrr genes within this region were significantly upregulated in the inoculated r treatment relative to the inoculated s treatment. rt-qpcr analysis of selected genes over the same r and s bulk samples showed a trend consistent with that of rpkm calculations and statistical analyses of transcript data. the rt-qpcr data confirmed up-regulation of a class- non-symbiotic hemoglobin, erd protein, protein tify 10a, s-adenosyl-l-methionine:carboxyl methyltransferase family protein, transcriptional factor bhlb92-like protein and transcriptional factor bhlh genes in resistant samples detected via rna-seq. the data also verified down-regulation of a chitinase-like protein, ralf-like protein, endochitinase isolog and cell-wall-protein-like protein genes as indicated in rna-seq analysis.figure validation of rpkm-calculated expression ratios for selected differentially expressed genes using rt-qpcr. rpkm values from rna-seq are denoted in black, and rt-qpcr results in while. capped lines represent the standard deviations from three biological replicates. annotation of degs the degs were functionally annotated based on go terms and sorted into the go-term biological process, molecular function and cellular component using blast2go. the statistics for go-term mapping were provided in additional file 1: figure s a total of degs retrieved no hits with blast. the annotated degs with upregulated patterns in r samples fell mainly into categories of biological process. the go term “defense response” was also one of the major categories identified. other upregulated biological processes of go terms included signal transduction, various metabolic/biosynthetic processes and regulation of metabolic processes. for molecular functions, several cellular-component go terms were identified, especially those associated with the plasma membrane representing the largest group. the go terms for biological processes associated with down-regulated degs were mostly in the category of “anatomical-structural and multicellular-organismal development” and “regulation of primary metabolic process”. most of the molecular functional go terms associated with down-regulated degs were in the same categories as those of upregulated degs, although several unique terms were identified, including sequence-specific dna binding transcription factor activity, hydrolase activity on o-glycosyl compounds, substrate-specific trans-membrane transporter activity, and nucleoside-triphosphatase activity. similarly, the majority of cellular-component go terms of down-regulated degs fell into categories similar to those of upregulated degs, with only three new go terms observed: vacuole, chloroplast stromal and organelle membrane.figure go terms associated with upregulated degs. a) go terms in the biological process category. b) go terms in the molecular functions. c) go terms in cellular components. the values labeled in the pie charts of panel a, b and c are the percentage of degs annotated with the corresponding go term relative to the total degs. go terms associated with down-regulated degs. a) go terms in the biological process category. b) go terms in molecular functions. c) go terms in cellular component. the values labeled in the pie charts of panel a, b and c are the percentage of degs annotated with the corresponding go term relative to the total degs. transcription factors were also characterized broadly for degs; a total of upregulated and down-regulated degs were grouped into seven types of tf, based on their conserved structures. for upregulated degs, of them belong to wrky, are myb domain-containing tfs, are ethylene -responsive tfs, are bhlh-domain containing tfs, belong to the ap2/erf family, are heat-stress related tfs, and the remaining belong to “other” tf families. for down-regulated degs, and of them fell into the respective tf families.table up-regulated differentially expressed genes annotated as transcription factors bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra013584 bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra001202 bra bra bra bra bra bra bra bra bra bra017656 bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra030208 bra bra bra bra bra bra bra bra bra bra bra bra bra011002 bra bra bra bra bra bra012828 bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra015582 bra bra bra bra bra bra bra bra030178 bra bra bra bra bra bra038774 bra bra002168 bra bra bra bra bra008716 bra bra bra bra bra bra bra bra008460 bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra bra014971 biological-process go terms for up- and down-regulated degs analysis using the fisher’s exact test in blast2go identified the enrichment associated with up- and down-regulated degs; a total of biological-process go terms displayed significant enrichment, with of them associated with upregulated and with down-regulated degs. most of these enriched go terms were related to “responses”, including those to chemical and hormone stimuli. the results were similar for metabolic-process go terms, with most of the enriched term being related to “responses”. among enriched go terms for upregulated degs, were related to lipid metabolism, including lipid metabolic process, cellular lipid metabolism, lipid biosynthesis, fatty acid metabolism, fatty acid biosynthesis, oxylipin metabolism and biosynthesis. several go terms related to defense-related phytohormones, including jasmonic acid and ethylene, but not salicylic acid, were also highly enriched in inoculated r plants relative to those in inoculated s plants. a total of degs were annotated under go terms with sa-related biological processes, including “response to sa stimulus”, “sa biosynthetic process “systemic acquired resistance, sa-mediated signaling pathway” etc., but none of them was significantly enriched in either up- or down-regulated go terms, as determined with the fisher’s exact test. noticeably, genes associated with several common defense responses were significantly upregulated, including “callose deposition”, “defense response by callose deposition” and “indole-containing compound metabolic process”. in contrast, most of the biological-process go terms enriched in down-regulated degs were related to “development” and “morphogenesis”, including developmental process, anatomical structure development, anatomical structure morphogenesis and uni-dimensional cell growth.figure comparison of go annotations of up- and down-regulated degs. blue bars represent the upregulated degs while red bars represent down-regulated degs. the values were the percentage of degs annotated with the corresponding go terms relative to the total up- or down-regulated degs. statistics of enrichment analysis are presented in the additional file 1: table s transcript analysis of selected defence-related degs using rt-qpcr based on rna-seq analysis, strongly upregulated and strongly down-regulated degs, involved possibly in resistance based on the functional annotation, were subjected to rt-qpcr analysis. all of the upregulated degs displayed similar transcriptional patterns; the transcript levels, based on their relative quantity, were comparable for r and s plants without pathogen inoculation but significantly higher in r plants after inoculation. the down-regulated degs, however, showed different transcriptional patterns; bra and bra displayed comparable transcript levels in non-inoculated s and r plants but these genes were significantly induced by p. brassicae in s plants and suppressed in r plants. bra and bra did not show significant induction by the pathogen in s plants relative to those in non-inoculated s or r plants, but suppressed in inoculated r plants. two of the tir-nbs-lrr genes residing in the rcr region were also identified as degs in the rna-seq, but were not included in the rt-qpcr test due to only moderate rpkm values. preliminary rt-qpcr trials on degs with moderate rpkm showed unsatisfactory amplification.figure the relative transcription quantity measured with rt-qpcr for selected upregulated degs identified in rna-seq. the treatments were s-susceptible and r-resistant plants inoculated with plasmodiophora brassicae or water. the vertical axis represents rtq against an endogenous control. treatments with one asterisk showed significantly higher rtq than those without asterisk, but lower rtq than those with two asterisks. the relative transcription quantity measured with rt-qpcr for selected down-regulated degs identified in rna-seq. the treatments were s-susceptible and r-resistant plants inoculated with plasmodiophora brassicae or water. the vertical axis represents rtq against an endogenous control. treatments with one asterisk had a significantly different level of rtq relative to without asterisk or with two asterisks. discussion mapping of the rcr1gene and development of genetic markers the cr gene rcr was mapped previously to a genomic region in the linkage group a flanked by the markers sn and sr6340i. additional markers were developed in the current study using the b. rapa genome information, with the markers a3- and ms7- being much closer to the cr gene than sr6340i. several markers were highly accurate in detecting rcr in both b. rapa and b. napus plants, especially when two flanking markers were used together. these markers will be useful for mas in resistance breeding. rcr is the only cr gene reported in pak choy, but the original source of the gene is not known. most of the cr genes identified in chinese cabbage originated from european turnip. four cr genes have been mapped previously to the linkage group a including cra, crb, crk and crr. cra has also been cloned recently. based on the relationship between common markers and the location of these cr loci, diederichsen et al. suggested that cra and crr are identical, allelic or at least closely linked to crb and crk. recent work also indicated that crb is likely in the same position as cra, and both genes conferred resistance to pathotypes and but not to pathotypes and of p. brassicae. based on the linkage distance, rcr is close to both cra and crb. however, the cv. fn which carries rcr is highly resistant to both pathotypes and, suggesting a different resistance spectrum from that of cra or crb. the mechanisms for clubroot resistance are not well understood. among the genes annotated within the region defined by sn and a3-, four tir-nbs-lrr genes can be located. the protein family containing nbs and lrr domains is the largest class of r genes cloned so far. cra and crr isolated from b. rapa also encode tir-nbs-lrr proteins. therefore, it is possible that rcr is one or a cluster of these tir-nbs-lrr genes in the mapped region. transcriptome analysis and go annotation of degs transcriptome profiling can provide insights into the mechanisms of disease resistance. this approach has been used to characterize several molecular components associated with clubroot disease development, especially the role of cytokinins on a. thaliana and b. juncea. however, no comparative analysis of gene transcription between clubroot resistant and susceptible plants was available. in our study, transcripts of genes were analyzed via rna-seq and degs were identified between inoculated r and s plants. this number of degs is comparable to that observed in a previous transcriptome analysis between rosette and folding leaves of chinese cabbage. fifty degs retrieved no hit in a blast search, indicating a number of unknown genes expressed in inoculated r plants. the go terms “defense response” and “plasma membrane” accounted for a substantial portion of upregulated degs. some of these genes may be candidates for further studies of clubroot resistance, because the plasma membrane is where most plant-pathogen interaction occurs. additionally, a large proportion of upregulated degs were annotated with the go term biological process involved in responses to external stimuli and many of these go terms were also significantly enriched for upregulated degs. these results indicate that many cellular activities in inoculated r plants were significantly upregulated at dpi when root infection and pathogen colonization were taking place. it also appears that infection may have caused differential activation or deactivation of certain genes in the host that may affect clubroot development on resistant and/or susceptible plants. the enrichment analysis identified go terms for biological process associated with both upregulated and down-regulated degs. several lipid compounds were implied to play a role in the inoculated r plants. lipids have been shown to play critical roles in detecting infection and may also have a key role in regulating gene transcription. during infection by p. brassicae, the r plants appeared to be able to mobilize lipid biosynthesis and metabolism, because several genes involved in oxylipin biosynthetic and metabolic processes, including bra bra and bra were upregulated substantially. one group of the most intensively studied oxylipins is jasmonates, likely due to their involvement in multiple plant biological processes. go terms associated with “response to jasmonic acid stimulus”, “ja metabolic process” and “ja biosynthetic process” were significantly enriched within the group of upregulated degs. this possibly indicates that jasmonates have a role in clubroot resistance mediated by rcr prost et al. employed in vitro growth inhibition assays to evaluate natural oxylipins and found that of them had inhibitory effects on a wide range of plant pathogens. similarly, jasmonates appear to play a role in induced resistance to clubroot caused by biofungicides. oxylipins may also be used by both host and pathogen as regulatory compounds during the host-pathogen interaction. further work is needed to confirm the specific role of oxylipins in clubroot resistance. potential molecular mechanisms for clubroot resistance several clubroot-resistance mechanisms identified previously were supported in the current study. for example, genes involved in olefin metabolic and biosynthetic processes and in response to et stimulus, were upregulated in plants carrying rcr. et had previously been shown to restrict club development in a. thaliana and to have a role in induced resistance mediated by biofungicides. this result supports the current opinion that ja and et may act synergistically in plant defense. reinforcement of plant cell wall was also indicated based on the enrichment of go terms for upregulated degs associated with callose localization and deposition. the role of callose has been well documented in resistance to penetration of plant cell wall by fungal pathogens. in arabidopsis, genes encoding the synthesis of β- glucan were suppressed in a compatible interaction between p. brassicae and susceptible ecotype col-. in the current study, the gene bra encoding an expansin-like protein, showed the highest increase in transcript among all degs upregulated. this may indicate a structural/composition alteration to the cell wall, which could affect secondary infection in epidermal and cortical cells. some of the above-mentioned defense-related degs were analyzed further with rt-qpcr using inoculated and non-inoculated plants to determine transcript levels as affected by p. brassicae. the results demonstrated that the inoculation increased the expression of these genes in both s and r plants. inoculated r plants, however, showed significantly stronger expression of these genes relative to inoculated s plants. this result is consistent with that of rna-seq. in addition, genes involved in synthesis of indole-containing compounds were also upregulated in r seedlings. the anti-microbial compounds derived from this metabolic process typically include tryptophan-derived metabolites and flavonoids converted from aromatic-acid phenylalanine. these indicate potential involvement of secondary metabolites in clubroot resistance. although several common resistance mechanisms are identified with rna-seq, further analysis is needed to assess the relative importance of each mechanism in clubroot resistance conferred by rcr as described above, rcr may encode a tir-nbs-lrr protein. the best characterized defense signaling pathway related to this class of r proteins involves the biosynthesis of salicylic acid and pathogenesis-related proteins, accompanied typically by a hypersensitive reaction. additionally, sa biosynthesis is linked frequently to host resistance against biotrophic pathogens. in the current study, however, go terms related to sa biosynthesis, metabolism or signaling processes were not significantly up-/down-regulated based on the enrichment analysis. this lack of substantial change in transcription of genes involved in sa biosynthesis has also been observed with induced resistance against clubroot on canola. previous reports have suggested hr as one of the possible mechanisms for clubroot resistance, but only two upregulated degs, i.e., bra and bra were identified as pr genes based on go annotation in the current study and there is no experimental evidence to link any of them to sa- or hr-related defense responses. in a cytological observation, deora et al. found no evidence of hr with clubroot resistant canola cultivars. it is possible that sa signaling pathways may not play a critical role in clubroot resistance. another mechanism for disease resistance is suppression of metabolic processes in the host that are required for pathogenesis. previous research had demonstrated that the level of auxin increased in roots during secondary infection by p. brassicae, likely as the result of enhanced biosynthesis and conversion of host auxin precursors induced by the pathogen. in the current study, bra which encodes a saur family of proteins, was among the most highly induced genes in r plants relative to s plants. saur proteins are closely linked to auxin biosynthesis and signaling. for example, saur had been found to be a negative regulator of auxin biosynthesis and transport in rice. with clubroot, pathogen-induced auxin metabolism had been linked to pathogenesis in arabidopsis. additionally, several genes encoding the auxin-responsive gh family of proteins were upregulated in r plants. the gh family has been linked with increased basal immunity via suppressing pathogen-induced auxin accumulation in rice. in addition to manipulating plant auxin homeostasis, over-expression of gh in an activation-tagged mutant of arabidopsis displayed enhanced biosynthesis of camalexin, the major phytoalexin found with pathogen infection in arabidopsis. since the biosynthesis of camalexin and auxin were derived from the common precursor tryptophan, the metabolic stream may have been redirected to flow into the biosynthesis of phytoalexin from auxin in the r plants. taken together, the results indicate that pathogen-activated auxin synthesis might have been suppressed or disrupted in r plants. most of the enriched go terms associated with down-regulated degs were related to growth and development, including cell cycle, uni-dimensional cell growth, anatomical structure morphogenesis, and cell morphogenesis. down-regulation of these genes may play a role in the resistance mediated by rcr because hypertrophy, the most typical symptom of clubroot, is related positively to these physiological activities. expression of genes involved in cell enlargement and proliferation was inhibited in an arabidopsis line partially resistant to p. brassicae, relative to the susceptible reaction. in the current study, two of the genes annotated for the go term “uni-dimensional cell growth” were upregulated by the pathogen in inoculated s plants, as opposed to the same genes in inoculated r plants that changed little relative to those in non-inoculated plants. down-regulation of these degs possibly works in conjunction with up-regulation of defense-related genes described above in resulting in a resistant outcome in plants carrying rcr it is not clear if down-regulation of growth/development degs is directly related to the suppression of auxin-dependent metabolism via enhanced expression of bra or gh genes observed. however, the evidence indicates that one or more processes related to auxin biosynthesis and cell growth/development are disrupted in r plants, with infection by p. brassicae. rna-seq analysis revealed that the clubroot resistance conferred by rcr involves complex mechanisms via a variety of biological processes controlled likely by corresponding transcription factors. as expected, upregulated degs were identified based on their conserved structures as tfs responsible for activation of several biological processes potentially involved in disease resistance. consistent with the roles of et and ja discussed above, et-responsive tfs including the ap2/erf-family tfs and bhlh-family tfs were upregulated to relatively high levels. wrky-family tfs were also upregulated. their involvement in disease resistance, including effector-triggered immunity, has been suggested previously. myb-family proteins may be involved in secondary metabolism including flavonoid and secondary cell-wall biosynthesis. these biological functions may contribute to clubroot resistance by generating anti-microbial metabolites and strengthening host cell walls. several heat-stress and “other” types of tfs were also identified, but their role in resistance is not understood. it is interesting that no heat-stress tfs were identified in the down-regulated degs, but the significance of this observation is unclear. only a small number of defense-related degs were present in the down-regulated degs sorted into the tf groups. the other down-regulated degs in tf groups are involved generally in cellular growth and development. in other words, these down-regulated tfs may represent targets that p. brassicae up-regulates during pathogenesis in s plants.  genetic resistance is the cornerstone for management of clubroot on canola. in this study, we characterized the cr gene rcr based on genetic mapping and transcript analysis, to develop markers for mas and decipher molecular mechanisms of resistance associated with rcr rna-seq analysis identified a range of biological processes potentially involved in clubroot resistance, consisting of both up-regulated defense-related and suppressed pathogenesis-related responses. this information is highly useful to design a breeding strategy based on modes of action of cr genes to achieve strong and durable clubroot resistance. although sa biosynthesis is often linked to plant resistance against biotrophic pathogens, genes involved in sa biosynthetic pathways were not activated in inoculated plants carrying rcr in contrast, genes involved in ja/et and callose biosynthesis were upregulated substantially. the biosynthesis or signaling of ja/et has not been identified previously for resistance to clubroot. further research is needed to confirm specific roles of these phytohormones in clubroot resistance mediated by rcr it will also be useful to look at these phytohormones in association with other cr genes. 
to identify new sources of clubroot resistance, we fine mapped a cr gene from b. rapa ssp. the identification of degs between inoculated plants carrying and not carrying rcr is an important step towards understanding of specific metabolic/signaling pathways in clubroot resistance mediated by rcr this information may help judicious use of cr genes with complementary resistance mechanisms for durable clubroot resistance. several degs involved in metabolism potentially related to clubroot symptom development, including auxin biosynthesis and cell growth/development, showed significantly lower expression in plants carrying rcr clubroot disease is a serious threat to the 8 m ha of canola grown annually in western canada. transcriptome analysis was conducted using rna sequencing on a segregating f population inoculated with p. brassicae, with differentially expressed genes identified between plants carrying and not carrying rcr functional annotation of these degs showed that several defense-related biological processes, including signaling and metabolism of jasmonate and ethylene, defensive deposition of callose and biosynthesis of indole-containing compounds, were up-regulated significantly in plants carrying rcr while genes involved in salicylic acid metabolic and signaling pathways were generally not elevated. while host resistance is the key to clubroot management, sources of resistance are limited. chinensis to the region between  mb and  mb on the linkage group a with several closely linked markers identified. the cr gene rcr and closely linked markers will be highly useful for breeding new resistant canola cultivars.
 the protist plasmodiophora brassicae is a biotrophic soil-borne pathogen that causes clubroot on brassica crops worldwide. clubroot disease is a serious threat to the 8 m ha of canola grown annually in western canada. while host resistance is the key to clubroot management, sources of resistance are limited.  to identify new sources of clubroot resistance, we fine mapped a cr gene from b. rapa ssp. chinensis to the region between  mb and  mb on the linkage group a with several closely linked markers identified. transcriptome analysis was conducted using rna sequencing on a segregating f population inoculated with p. brassicae, with differentially expressed genes identified between plants carrying and not carrying rcr functional annotation of these degs showed that several defense-related biological processes, including signaling and metabolism of jasmonate and ethylene, defensive deposition of callose and biosynthesis of indole-containing compounds, were up-regulated significantly in plants carrying rcr while genes involved in salicylic acid metabolic and signaling pathways were generally not elevated. several degs involved in metabolism potentially related to clubroot symptom development, including auxin biosynthesis and cell growth/development, showed significantly lower expression in plants carrying rcr  the cr gene rcr and closely linked markers will be highly useful for breeding new resistant canola cultivars. the identification of degs between inoculated plants carrying and not carrying rcr is an important step towards understanding of specific metabolic/signaling pathways in clubroot resistance mediated by rcr this information may help judicious use of cr genes with complementary resistance mechanisms for durable clubroot resistance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords clubrootplasmodiophora brassicaegenetic mappingmarker-assisted selectionnext-generation sequencingrna-seqgene ontologytranscription factorsissue-copyright-statement© the author 2014 