BACKGROUND
when plants are under attack from fungal pathogens, they can often detect the secretion of fungal effector proteins, either directly or indirectly, by means of plant resistance  genes, which initiate a defense response known as effector-triggered immunity .
eti often causes localized cell death described as a hypersensitive response  and prevents further infection  <cit> .
r-genes are of vital importance in providing protection from plant pathogens, and an understanding of the relationship between race-specific r-genes and their corresponding pathogen avirulence  genes  <cit>  is required for the effective deployment of resistance genetics in crop varieties.
the hemibiotrophic fungal pathogen leptosphaeria maculans  ces.
& de not.
desmaz.)
is the causal agent of blackleg disease; the most economically important disease of brassica crops worldwide  <cit> .
eighteen major r-genes for blackleg disease have been identified in brassica species, though several of these are probably redundant .
most of the r-genes effective against blackleg described to date map to one of two chromosomes in the brassica a genome; rlm <dig>  rlm <dig>  rlm <dig>  rlm <dig> and rlm <dig> form a cluster of r-genes on chromosome a <dig>  <cit> , while lepr <dig>  <cit> , blmr2/rlms  lepr <dig>  <cit>  and rlm <dig>  <cit>  map to chromosome a <dig>  rlm <dig> was identified from the european brassica napus  variety ‘glacier’  <cit> , which was later shown to contain two r-genes; rlm <dig> and rlm <dig>  <cit> .
lepr <dig> was identified from the b. napus cultivar “surpass 400”  <cit>  and was reportedly introgressed into b. napus from b. rapa subsp.
sylvestris  <cit> .
lepr <dig> is to date the only r-gene for resistance to blackleg to be cloned.
it encodes a receptor-like protein  which conveys resistance via hr during infection by l. maculans isolates expressing the avrlm <dig> avirulence gene  <cit> .
the gene ‘bnaa10g20720d’ on chromosome a <dig> of the newly-sequenced b. napus reference ‘darmor-bzh’ genome  <cit> , homologous to the b. rapa gene bra <dig>  <cit> , shares 99% identity with the susceptible lepr <dig> allele of ‘topas dh16516’  <cit> .
rlm <dig> confers race-specific resistance to l. maculans isolates harbouring the corresponding avirulence gene avrlm <dig>  <cit> , which forms part of a genetic cluster of avirulence genes  within the l. maculans genome  <cit> .
previously, the map location of rlm <dig> was investigated using two resistant cultivars; ‘glacier’ and ‘samouraï’, and shown to be positioned in the same interval of lg <dig> , confirming the same gene was present in both varieties.
the gene was positioned within an interval of 12 cm on the ‘darmor’ x ‘samouraï’ lg <dig>  map  <cit> , though the lack of genomic resources available at the time prevented physical definition of the gene interval.
rlm <dig> has been detected in many other european winter-type canola varieties, including one of the first blackleg-resistant cultivars ‘ramsès’  <cit> , which was also used in the development of early australian blackleg-resistant varieties  <cit> .
rlm <dig> has also been detected in b. rapa  <cit> .
the aim of the current study was to define the precise map location of rlm <dig>  and identify tightly-linked markers for use in marker-assisted breeding programs.
by aligning the b. napus map interval containing rlm <dig> to the dna sequence of b. rapa and b. napus for the corresponding region of chromosome a <dig>  <cit> , we were able to physically define the interval containing the rlm <dig> locus on chromosome a <dig>  determine the position of rlm <dig> relative to lepr <dig> and identify several candidate defense-related gene homologues.
RESULTS
phenotypic analysis
the l. maculans isolate ‘165’ was used to inoculate  <dig> seedlings of each parental line used to construct the bc1f <dig> population.
infection with the isolate resulted in test scores between  <dig> and  <dig> on ‘topas’  and scores of  <dig> to  <dig> on ‘glacier dh24287’ .
further tests using additional control b. napus lines showed ‘165’ was avirulent on the rlm <dig> line ‘tapidor dh’  and virulent on the rlm <dig> variety ‘quantum’ , the rlm1/rlm <dig> variety ‘columbus’ , ‘topas dh16516’  and the topas dh16516:lepr <dig> transgenic line ‘nla8-2’ .
these results confirmed the “avrlm <dig>  avrlm <dig>  avrlm3” pathotype of the isolate .figure  <dig>
interaction of
l. maculans
isolate ‘165’ with differential lines of
b. napus
.
cotyledons of seven b. napus lines pictured at 14-day post-inoculation.
lines containing rlm <dig>  showed typical hypersensitive response and restriction of lesions while lines absent rlm <dig>  were fully susceptible to infection.
phenotypic screening of the  <dig> bc1f <dig>  individuals showed segregation for the rlm <dig> phenotype, with  <dig> seedlings scored as resistant to ‘165’  and  <dig> seedlings scored as susceptible .
this conformed to a 1: <dig> ratio  as expected for a single dominant gene.
rlm <dig> mapping
twenty microsatellite markers spanning chromosome a <dig> were screened and a set of four markers  that closely segregated with the rlm <dig> locus were selected for genotyping the topas x glacier dh <dig> bc1f <dig> population.
these markers spanned the equivalent of  <dig> genes  and a physical interval of approximately 1 mb of the b. rapa genome  <cit> .
the selected microsatellite markers were used in conjunction with the initial  <dig> bc1f <dig> individuals to produce a draft map of the rlm <dig> interval using mapmaker v <dig> b software  <cit>  in order to confirm their linkage to the phenotype  and that they flanked the rlm <dig> locus.
the remaining bc1f <dig> individuals were then screened for recombination within the rlm <dig> region.
a total of  <dig> bc1f <dig> individuals where successfully genotyped with the flanking microsatellite markers.
markers were re-run on the putative recombinant bc1f <dig> individuals and any non-confirmed recombinants discarded.
after rescreening and phenotyping of the bc1f <dig> generation a total of  <dig> confirmed recombinants were retained for the informative recombinant mapping subset, each containing a single recombination event within the map interval.
the resulting map of ‘topas’ x ‘glacier’ a <dig>  showed rlm <dig> was contained within an interval of  <dig>  cm, between sn <dig> and sn <dig> .
this interval corresponded to a collinear span of approximately 926 kb of the b. rapa genome, containing  <dig> putative genes on chromosome a <dig> .
the majority of the cross-overs detected in the population  occurred either between sn <dig> and the rlm <dig> locus , or between the rlm <dig> locus and sn <dig> .
two additional markers were designed for the rlm <dig> interval; one sequence characterised amplified region  marker , targeted to the bra <dig> homologue  and one cleaved amplified polymorphic sequence  marker , targeted to the bra <dig> homologue.
ind10- <dig> produced fragments of 142 bp for ‘topas’ and 138 bp for ‘glacier’.
caps <dig> produces amplicons of approximately 900 bp from both parents.
digestion with bstui produces fragments of  <dig> and 410 bp from the ‘glacier’ a-genome amplicon only.
when used to genotype the recombinant subset of the mapping population, both of these markers co-segregated with the rlm <dig> phenotype, providing markers tightly-linked to the rlm <dig> gene yet failing to reduce the size of the target map interval.
an additional nine microsatellite markers positioned within the map interval were tested but also failed to provide additional informative data.figure  <dig>
comparison of genetic maps for
rlm2
and
lepr3
to
b. napus
chromosome a <dig>  position of rlm <dig> relative to microsatellite , indel  and caps markers on ‘topas’ x ‘glacier’ a <dig>  map.
nearest b. napus gene to each marker given in brackets.
unanchored b. napus genes corresponding to syntenic b. rapa a <dig> homologues are denoted with “*”.
lepr <dig> ‘topas’ x ‘surpass 400’ a <dig>  map updated from larkan et al., 2013a.
marker intervals given in centimorgans.
solid horizontal lines denote same marker, dashed horizontal lines denote markers sharing same nearest b. napus homologue.
approximate physical location of defense-related candidate genes by triangles.
gap in b. napus a <dig> chromosome build represented by dotted lines.
comparison of the rlm <dig> and lepr <dig> maps showed that both genes were located within the same genetic interval on chromosome a <dig>  while one additional marker was integrated from the lepr <dig> map , this also co-segregated with the rlm <dig> phenotype.
all remaining markers used in the lepr <dig> map were non-polymorphic in the ‘topas’ x ‘glacier’ population.
the maps share three common markers and two other markers that have a common closest b. napus gene, and the cluster of markers that co-segregate with rlm <dig> span the lepr <dig> locus .
candidate gene selection
inspection of the  <dig> genes contained within the region of b. rapa a <dig> collinear to the rlm <dig> map interval lead to the identification of seven candidate gene homologues, selected on the basis of their potential roles in resistance to microbial pathogens.
two of the candidates  are homologous to a. thaliana genes involved in disease resistance signaling during infection by the bacterial pathogen pseudomonas syringae  <cit> .
the interval also contained bra <dig>  previously identified as the b. rapa homologue of the b. napus blackleg resistance gene lepr <dig>  <cit> , and four other genes  of unknown function in a. thaliana that are members of gene families involved in plant resistance responses .
two of these, members of the leucine rich-repeat  protein family  were examined using interpro  <dig> and lrrfinder  <dig>  and were both predicted to encode small  proteins with predicted primary structures featuring signal peptides, lrr n-terminal domains, seven extracellular lrr domains and potential c-terminal lrr domains, similar to members of the plant defense-related polygalacturonase inhibitor protein  family  <cit> .
blast analysis showed these proteins shared 60% identity with pgip-like family members from poplar .table  <dig>
candidate defense-related
b. napus
genes identified within a <dig> region corresponding to the
rlm2
map interval
b. napus
gene
a. thaliana
homologue
a. thaliana
description
role
genes denoted with “*” are not anchored to b. napus a <dig> yet correspond to syntenic b. rapa a <dig> homologues.
alignment of the rlm <dig> map interval to the b. napus after the release of the ‘darmor-bzh’ reference genome  <cit>  revealed a physical interval of approximately 873 kb spanning  <dig> genes  containing syntenic homologues of  <dig> of the  <dig> candidate genes previously identified in b. rapa.
the two ‘missing’ b. rapa homologues  had been assigned to an unanchored “chra10_random” pseudo-molecule, with the collinear b. napus a <dig> region spanning the genes in b. rapa being represented by a gap of approximately 43 kb .
discussion
we were able to define the rlm <dig> locus to a map interval of  <dig>  cm and a physical interval of  <dig> predicted b. napus genes, though further dissection of the rlm <dig> interval was hampered by a lack of polymorphism, as many additional markers located within the region proved to be monomorphic in our population.
we could make efforts to produce additional markers targeted to the interval, however the recent advent of high-density marker systems for b. napus, such as dart  <cit> , illumina infinium 6 k  <cit>  or 60 k  snp arrays, or genotyping-by-sequencing methods  <cit>  makes the pursuit of further specific sequence-characterised markers impractical.
while we have provided several markers tightly-linked to the rlm <dig> locus which could be of use in blackleg resistance breeding programs, the real value of the work is in defining the physical location of rlm <dig> on chromosome a <dig>  with this information any sequence-characterised marker set, including the high-density systems, can be orientated to the map in order to identify markers linked to the physical location of the gene.
the co-localisation of the blackleg resistance genes lepr <dig> and rlm <dig> in the b. napus genome reported here draws an interesting comparison to the clustering of their corresponding avirulence genes, avrlm <dig> and avrlm <dig>  in the l. maculans genome  <cit> .
molecular characterisation of rlm <dig> and avrlm <dig> and comparison to the previously-identified lepr <dig>  <cit>  and avrlm <dig>  <cit>  may provide some insight into the evolution of the molecular ‘arms race’ between plant and pathogen.
we identified seven potential resistance-related homologues in the regions of b. rapa and b. napus genomes corresponding to the map interval containing rlm <dig> .
one of these genes, bnaa10g20720d, corresponds to the lepr <dig> blackleg-resistance locus and was targeted in this study by the scar marker ind10- <dig>  this marker co-segregated with the resistance phenotype, as did two other markers corresponding to neighbouring genes .
this suggests that rlm <dig> is located close to this region of the chromosome however, based on our results, we cannot rule out other candidate genes within the wider map interval .
these include a member of the calcium-dependant protein kinase  family .
cpks have been shown to be positive regulators of race-specific pathogen defense in several plant species  <cit> .
they act to facilitate hr during eti-mediated defense responses after translocation from the cytosol to the nucleus, where they activate wrky transcription factors  <cit> .
other candidates include a member of the tir-nbs-lrr class of resistance genes , which are well established as initiators of plant resistance responses during both direct and indirect interactions with pathogen effectors  <cit> , and two genes  homologous to a. thaliana genes pbs <dig> and pbs <dig>  respectively, involved in the p. syringae resistance response  <cit> .
pbs <dig> encodes a receptor-like cytoplasmic kinase targeted for cleavage by the p. syringae effector avrpphb, which triggers the cc-nbs-lrr class resistance protein rps <dig>  <cit> .
the final two candidate genes, bnaa10g30070d and bnaa10g30080d, encode putative pgip-like proteins.
pgips are small lrr-containing proteins that are secreted from the host cell into the apoplastic fluid where they inhibit host cell wall degradation by binding to pathogen endopolygalacturonases during infection by fungi  <cit> , nematodes  <cit>  and other plant pathogens.
though bnaa10g30070d and bnaa10g30080d were not incorporated in the main a <dig> chromosome build of the initial b. napus genome release, their b. rapa  and a. thaliana  homologues are located in regions syntenic to the rlm <dig> map interval in their respective genomes  <cit> .
the genomic interval containing rlm <dig> in the b. napus variety ‘samouraï’  <cit>  was also shown to harbour a qtl for l. maculans resistance during field trials in france  <cit>  despite the absence of the corresponding avirulence gene avrlm <dig> in french isolates  <cit> .
it was suggested that either rlm <dig> has a residual effect on avrlm <dig> l. maculans isolates, or that another genetic factor limiting growth of the pathogen was linked to the rlm <dig> locus  <cit> .
a number of the candidate defense-related genes identified here as being linked to the rlm <dig> locus could also be of interest as candidates in non-race specific adult-plant resistance.
in particular, the interaction of plant pgips with pathogen polygalacturonases inhibits the degradation of the host cell wall and can also lead to accumulation of non-specific defense responses such as lignification and the production of reactive oxygen species  <cit> .
fungal polygalacturonases have been shown to accumulate in b. napus stems during infection by l. maculans and may play an important role in the development of canker lesions  <cit>  though expression of the l. maculans polygalacturonase-encoding pg <dig> gene was not detected during cotyledon and leave infection  <cit> .
if the candidate genes we identified do indeed encode functional pgip proteins then they could potentially play a role in the suppression of blackleg disease in the adult plant.
the detailed genetic map and physical location of the rlm <dig> gene presented here should aid in the marker-assisted breeding of the gene into modern b. napus varieties.
while rlm <dig> is of little use in europe, due to the absence of the matching avrlm <dig> avirulence gene in most european l. maculans populations  <cit> , it is a valuable source of resistance to blackleg disease in canada.
a survey of western canadian isolates showed that 100% of isolates collected between  <dig> and  <dig>  and  <dig> % of isolates collected between  <dig> and  <dig>  harboured avrlm <dig>  this high frequency was unexpected, as rlm <dig> has been available to canadian plant breeders for some time and a greater adaptation by the pathogen was expected  <cit>  suggesting rlm <dig> has not yet been widely deployed in canadian b. napus varieties.
while rlm <dig> is potentially valuable in developing blackleg resistance canola varieties in north america, breeders must be cautious in its deployment.
the lack of avrlm <dig> in current european l. maculans populations stands in stark contrast to the effectiveness of rlm <dig> in controlling blackleg in europe several decades ago.
high selection pressure has resulted in a dynamic evolution of race structure within the pathogen populations of europe, leading to the sequential loss of rlm <dig>  rlm <dig> and rlm1-mediated resistance  <cit> .
a similar situation could develop in north america; an earlier survey of l. maculans isolates in southern ontario, a winter-type canola growing region geographically isolated from the prairies of western canada, found no avrlm <dig> isolates  <cit> .
kutcher et al.
noted that the only avrlm <dig> isolates detected in their survey were collected in southern manitoba in 2003– <dig>  another survey also found avrlm <dig> isolates both in alberta, manitoba and directly south across the canada/us border in north dakota  <cit>  and a recent report suggests nearly all l. maculans in north dakota would be virulent on rlm <dig> varieties  <cit> .
clearly relying on rlm <dig> as the single resistance source for a canola variety would be foolhardy.
proper stewardship of the gene would entail pyramiding the resistance by combining rlm <dig> with other effective r-genes and/or quantitative resistance genetics  <cit> .
CONCLUSIONS
we are presented with three scenarios as to the identity of rlm2; 1) the co-localisation of rlm <dig> and lepr <dig> is due to the genes being allelic variants of the same gene locus , 2) rlm <dig> corresponds to one of the other b. napus candidate defense-related homologues identified within the syntenic map interval, or 3) rlm <dig> is a gene specific only to certain varieties of b. napus and not represented in the b. napus ‘darmor-bzh’ or b. rapa var.
‘chiifu’ genome sequences.
investigation of the candidate genes identified in this study is currently underway.
regardless of the molecular identity of rlm <dig>  delimiting the physical region of the b. napus genome that harbours the gene provides the information required for the efficient marker-assisted selection of rlm <dig> in modern canola breeding programs.

 when plants are under attack from fungal pathogens, they can often detect the secretion of fungal effector proteins, either directly or indirectly, by means of plant resistance genes, which initiate a defense response known as effector-triggered immunity. eti often causes localized cell death described as a hypersensitive response and prevents further infection. r-genes are of vital importance in providing protection from plant pathogens, and an understanding of the relationship between race-specific r-genes and their corresponding pathogen avirulence genes is required for the effective deployment of resistance genetics in crop varieties. the hemibiotrophic fungal pathogen leptosphaeria maculans ces. & de not. desmaz.) is the causal agent of blackleg disease; the most economically important disease of brassica crops worldwide. eighteen major r-genes for blackleg disease have been identified in brassica species, though several of these are probably redundant. most of the r-genes effective against blackleg described to date map to one of two chromosomes in the brassica a genome; rlm rlm rlm rlm and rlm form a cluster of r-genes on chromosome a, while lepr, blmr2/rlms lepr and rlm map to chromosome a rlm was identified from the european brassica napus variety ‘glacier’, which was later shown to contain two r-genes; rlm and rlm. lepr was identified from the b. napus cultivar “surpass 400” and was reportedly introgressed into b. napus from b. rapa subsp. sylvestris. lepr is to date the only r-gene for resistance to blackleg to be cloned. it encodes a receptor-like protein which conveys resistance via hr during infection by l. maculans isolates expressing the avrlm avirulence gene. the gene ‘bnaa10g20720d’ on chromosome a of the newly-sequenced b. napus reference ‘darmor-bzh’ genome, homologous to the b. rapa gene bra, shares 99% identity with the susceptible lepr allele of ‘topas dh16516’. rlm confers race-specific resistance to l. maculans isolates harbouring the corresponding avirulence gene avrlm, which forms part of a genetic cluster of avirulence genes within the l. maculans genome. previously, the map location of rlm was investigated using two resistant cultivars; ‘glacier’ and ‘samouraï’, and shown to be positioned in the same interval of lg, confirming the same gene was present in both varieties. the gene was positioned within an interval of 12 cm on the ‘darmor’ x ‘samouraï’ lg map, though the lack of genomic resources available at the time prevented physical definition of the gene interval. rlm has been detected in many other european winter-type canola varieties, including one of the first blackleg-resistant cultivars ‘ramsès’, which was also used in the development of early australian blackleg-resistant varieties. rlm has also been detected in b. rapa. the aim of the current study was to define the precise map location of rlm and identify tightly-linked markers for use in marker-assisted breeding programs. by aligning the b. napus map interval containing rlm to the dna sequence of b. rapa and b. napus for the corresponding region of chromosome a, we were able to physically define the interval containing the rlm locus on chromosome a determine the position of rlm relative to lepr and identify several candidate defense-related gene homologues.  phenotypic analysis the l. maculans isolate ‘165’ was used to inoculate seedlings of each parental line used to construct the bc1f population. infection with the isolate resulted in test scores between and on ‘topas’ and scores of to on ‘glacier dh24287’. further tests using additional control b. napus lines showed ‘165’ was avirulent on the rlm line ‘tapidor dh’ and virulent on the rlm variety ‘quantum’, the rlm1/rlm variety ‘columbus’, ‘topas dh16516’ and the topas dh16516:lepr transgenic line ‘nla8-2’. these results confirmed the “avrlm avrlm avrlm3” pathotype of the isolate.figure interaction of l. maculans isolate ‘165’ with differential lines of b. napus. cotyledons of seven b. napus lines pictured at 14-day post-inoculation. lines containing rlm showed typical hypersensitive response and restriction of lesions while lines absent rlm were fully susceptible to infection. phenotypic screening of the bc1f individuals showed segregation for the rlm phenotype, with seedlings scored as resistant to ‘165’ and seedlings scored as susceptible. this conformed to a 1: ratio as expected for a single dominant gene. rlm mapping twenty microsatellite markers spanning chromosome a were screened and a set of four markers that closely segregated with the rlm locus were selected for genotyping the topas x glacier dh bc1f population. these markers spanned the equivalent of genes and a physical interval of approximately 1 mb of the b. rapa genome. the selected microsatellite markers were used in conjunction with the initial bc1f individuals to produce a draft map of the rlm interval using mapmaker v b software in order to confirm their linkage to the phenotype and that they flanked the rlm locus. the remaining bc1f individuals were then screened for recombination within the rlm region. a total of bc1f individuals where successfully genotyped with the flanking microsatellite markers. markers were re-run on the putative recombinant bc1f individuals and any non-confirmed recombinants discarded. after rescreening and phenotyping of the bc1f generation a total of confirmed recombinants were retained for the informative recombinant mapping subset, each containing a single recombination event within the map interval. the resulting map of ‘topas’ x ‘glacier’ a showed rlm was contained within an interval of  cm, between sn and sn. this interval corresponded to a collinear span of approximately 926 kb of the b. rapa genome, containing putative genes on chromosome a. the majority of the cross-overs detected in the population occurred either between sn and the rlm locus, or between the rlm locus and sn. two additional markers were designed for the rlm interval; one sequence characterised amplified region marker, targeted to the bra homologue and one cleaved amplified polymorphic sequence marker, targeted to the bra homologue. ind10- produced fragments of 142 bp for ‘topas’ and 138 bp for ‘glacier’. caps produces amplicons of approximately 900 bp from both parents. digestion with bstui produces fragments of and 410 bp from the ‘glacier’ a-genome amplicon only. when used to genotype the recombinant subset of the mapping population, both of these markers co-segregated with the rlm phenotype, providing markers tightly-linked to the rlm gene yet failing to reduce the size of the target map interval. an additional nine microsatellite markers positioned within the map interval were tested but also failed to provide additional informative data.figure comparison of genetic maps for rlm2 and lepr3 to b. napus chromosome a position of rlm relative to microsatellite, indel and caps markers on ‘topas’ x ‘glacier’ a map. nearest b. napus gene to each marker given in brackets. unanchored b. napus genes corresponding to syntenic b. rapa a homologues are denoted with “*”. lepr ‘topas’ x ‘surpass 400’ a map updated from larkan et al., 2013a. marker intervals given in centimorgans. solid horizontal lines denote same marker, dashed horizontal lines denote markers sharing same nearest b. napus homologue. approximate physical location of defense-related candidate genes by triangles. gap in b. napus a chromosome build represented by dotted lines. comparison of the rlm and lepr maps showed that both genes were located within the same genetic interval on chromosome a while one additional marker was integrated from the lepr map, this also co-segregated with the rlm phenotype. all remaining markers used in the lepr map were non-polymorphic in the ‘topas’ x ‘glacier’ population. the maps share three common markers and two other markers that have a common closest b. napus gene, and the cluster of markers that co-segregate with rlm span the lepr locus. candidate gene selection inspection of the genes contained within the region of b. rapa a collinear to the rlm map interval lead to the identification of seven candidate gene homologues, selected on the basis of their potential roles in resistance to microbial pathogens. two of the candidates are homologous to a. thaliana genes involved in disease resistance signaling during infection by the bacterial pathogen pseudomonas syringae. the interval also contained bra previously identified as the b. rapa homologue of the b. napus blackleg resistance gene lepr, and four other genes of unknown function in a. thaliana that are members of gene families involved in plant resistance responses. two of these, members of the leucine rich-repeat protein family were examined using interpro and lrrfinder and were both predicted to encode small proteins with predicted primary structures featuring signal peptides, lrr n-terminal domains, seven extracellular lrr domains and potential c-terminal lrr domains, similar to members of the plant defense-related polygalacturonase inhibitor protein family. blast analysis showed these proteins shared 60% identity with pgip-like family members from poplar.table candidate defense-related b. napus genes identified within a region corresponding to the rlm2 map interval b. napus gene a. thaliana homologue a. thaliana description role genes denoted with “*” are not anchored to b. napus a yet correspond to syntenic b. rapa a homologues. alignment of the rlm map interval to the b. napus after the release of the ‘darmor-bzh’ reference genome revealed a physical interval of approximately 873 kb spanning genes containing syntenic homologues of of the candidate genes previously identified in b. rapa. the two ‘missing’ b. rapa homologues had been assigned to an unanchored “chra10_random” pseudo-molecule, with the collinear b. napus a region spanning the genes in b. rapa being represented by a gap of approximately 43 kb. discussion we were able to define the rlm locus to a map interval of  cm and a physical interval of predicted b. napus genes, though further dissection of the rlm interval was hampered by a lack of polymorphism, as many additional markers located within the region proved to be monomorphic in our population. we could make efforts to produce additional markers targeted to the interval, however the recent advent of high-density marker systems for b. napus, such as dart, illumina infinium 6 k or 60 k snp arrays, or genotyping-by-sequencing methods makes the pursuit of further specific sequence-characterised markers impractical. while we have provided several markers tightly-linked to the rlm locus which could be of use in blackleg resistance breeding programs, the real value of the work is in defining the physical location of rlm on chromosome a with this information any sequence-characterised marker set, including the high-density systems, can be orientated to the map in order to identify markers linked to the physical location of the gene. the co-localisation of the blackleg resistance genes lepr and rlm in the b. napus genome reported here draws an interesting comparison to the clustering of their corresponding avirulence genes, avrlm and avrlm in the l. maculans genome. molecular characterisation of rlm and avrlm and comparison to the previously-identified lepr and avrlm may provide some insight into the evolution of the molecular ‘arms race’ between plant and pathogen. we identified seven potential resistance-related homologues in the regions of b. rapa and b. napus genomes corresponding to the map interval containing rlm. one of these genes, bnaa10g20720d, corresponds to the lepr blackleg-resistance locus and was targeted in this study by the scar marker ind10- this marker co-segregated with the resistance phenotype, as did two other markers corresponding to neighbouring genes. this suggests that rlm is located close to this region of the chromosome however, based on our results, we cannot rule out other candidate genes within the wider map interval. these include a member of the calcium-dependant protein kinase family. cpks have been shown to be positive regulators of race-specific pathogen defense in several plant species. they act to facilitate hr during eti-mediated defense responses after translocation from the cytosol to the nucleus, where they activate wrky transcription factors. other candidates include a member of the tir-nbs-lrr class of resistance genes, which are well established as initiators of plant resistance responses during both direct and indirect interactions with pathogen effectors, and two genes homologous to a. thaliana genes pbs and pbs respectively, involved in the p. syringae resistance response. pbs encodes a receptor-like cytoplasmic kinase targeted for cleavage by the p. syringae effector avrpphb, which triggers the cc-nbs-lrr class resistance protein rps. the final two candidate genes, bnaa10g30070d and bnaa10g30080d, encode putative pgip-like proteins. pgips are small lrr-containing proteins that are secreted from the host cell into the apoplastic fluid where they inhibit host cell wall degradation by binding to pathogen endopolygalacturonases during infection by fungi, nematodes and other plant pathogens. though bnaa10g30070d and bnaa10g30080d were not incorporated in the main a chromosome build of the initial b. napus genome release, their b. rapa and a. thaliana homologues are located in regions syntenic to the rlm map interval in their respective genomes. the genomic interval containing rlm in the b. napus variety ‘samouraï’ was also shown to harbour a qtl for l. maculans resistance during field trials in france despite the absence of the corresponding avirulence gene avrlm in french isolates. it was suggested that either rlm has a residual effect on avrlm l. maculans isolates, or that another genetic factor limiting growth of the pathogen was linked to the rlm locus. a number of the candidate defense-related genes identified here as being linked to the rlm locus could also be of interest as candidates in non-race specific adult-plant resistance. in particular, the interaction of plant pgips with pathogen polygalacturonases inhibits the degradation of the host cell wall and can also lead to accumulation of non-specific defense responses such as lignification and the production of reactive oxygen species. fungal polygalacturonases have been shown to accumulate in b. napus stems during infection by l. maculans and may play an important role in the development of canker lesions though expression of the l. maculans polygalacturonase-encoding pg gene was not detected during cotyledon and leave infection. if the candidate genes we identified do indeed encode functional pgip proteins then they could potentially play a role in the suppression of blackleg disease in the adult plant. the detailed genetic map and physical location of the rlm gene presented here should aid in the marker-assisted breeding of the gene into modern b. napus varieties. while rlm is of little use in europe, due to the absence of the matching avrlm avirulence gene in most european l. maculans populations, it is a valuable source of resistance to blackleg disease in canada. a survey of western canadian isolates showed that 100% of isolates collected between and and % of isolates collected between and harboured avrlm this high frequency was unexpected, as rlm has been available to canadian plant breeders for some time and a greater adaptation by the pathogen was expected suggesting rlm has not yet been widely deployed in canadian b. napus varieties. while rlm is potentially valuable in developing blackleg resistance canola varieties in north america, breeders must be cautious in its deployment. the lack of avrlm in current european l. maculans populations stands in stark contrast to the effectiveness of rlm in controlling blackleg in europe several decades ago. high selection pressure has resulted in a dynamic evolution of race structure within the pathogen populations of europe, leading to the sequential loss of rlm rlm and rlm1-mediated resistance. a similar situation could develop in north america; an earlier survey of l. maculans isolates in southern ontario, a winter-type canola growing region geographically isolated from the prairies of western canada, found no avrlm isolates. kutcher et al. noted that the only avrlm isolates detected in their survey were collected in southern manitoba in 2003– another survey also found avrlm isolates both in alberta, manitoba and directly south across the canada/us border in north dakota and a recent report suggests nearly all l. maculans in north dakota would be virulent on rlm varieties. clearly relying on rlm as the single resistance source for a canola variety would be foolhardy. proper stewardship of the gene would entail pyramiding the resistance by combining rlm with other effective r-genes and/or quantitative resistance genetics.  we are presented with three scenarios as to the identity of rlm2; 1) the co-localisation of rlm and lepr is due to the genes being allelic variants of the same gene locus, 2) rlm corresponds to one of the other b. napus candidate defense-related homologues identified within the syntenic map interval, or 3) rlm is a gene specific only to certain varieties of b. napus and not represented in the b. napus ‘darmor-bzh’ or b. rapa var. ‘chiifu’ genome sequences. investigation of the candidate genes identified in this study is currently underway. regardless of the molecular identity of rlm delimiting the physical region of the b. napus genome that harbours the gene provides the information required for the efficient marker-assisted selection of rlm in modern canola breeding programs. 
in this study, the rlm gene for resistance to blackleg, located on chromosome a of the b. napus cultivar ‘glacier’, was targeted for fine mapping. molecular markers tightly linked to the gene were developed for use in mapping the resistance locus and defining the physical interval in b. napus. rlm was localised to a  cm interval corresponding to approximately 873 kb of the b. napus chromosome a the recently-cloned b. napus r-gene, lepr occupies the same region of a as rlm and analysis of the putative b. napus and b. rapa genes in the homologous region identified several additional candidate defense-related genes that may control rlm function. while many r-genes effective against blackleg disease have been identified in brassica species, information of the precise genomic locations of the genes is limited. the protection of canola crops against blackleg disease, caused by the fungal pathogen leptosphaeria maculans, is largely mediated by race-specific resistance genes.
 the protection of canola crops against blackleg disease, caused by the fungal pathogen leptosphaeria maculans, is largely mediated by race-specific resistance genes. while many r-genes effective against blackleg disease have been identified in brassica species, information of the precise genomic locations of the genes is limited.  in this study, the rlm gene for resistance to blackleg, located on chromosome a of the b. napus cultivar ‘glacier’, was targeted for fine mapping. molecular markers tightly linked to the gene were developed for use in mapping the resistance locus and defining the physical interval in b. napus. rlm was localised to a  cm interval corresponding to approximately 873 kb of the b. napus chromosome a  the recently-cloned b. napus r-gene, lepr occupies the same region of a as rlm and analysis of the putative b. napus and b. rapa genes in the homologous region identified several additional candidate defense-related genes that may control rlm function. keywords blacklegbrassica napusleptosphaeria maculansmarker-assisted breedingmolecular markerpgip-like proteinreceptor-like proteinresistanceissue-copyright-statement© the author 2014 