BACKGROUND
phoma medicaginis is the causal agent of spring black stem and leaf spot in alfalfa , a major fodder and forage crop in temperate and mediterranean regions.
studies in four north-eastern us states have shown foliar necrotrophs, principally p. medicaginis, resulted in estimated average yield losses of over 13%.
for harvests where significant yield losses occurred, this figure rose to over 19%  <cit> .
p. medicaginis also causes disease on the model legume medicago truncatula, or barrel medic, which is used in australia in ley rotations to enhance soil nitrogen  <cit> .
in susceptible cultivars, reduction in seed and herbage yields, and almost complete defoliation and premature death has been reported  <cit> , with infected cultivars exhibiting an average seed weight reduction of  <dig> %  <cit> .
chemical and cultural control of foliar necrotrophs like p. medicaginis has proved to be expensive and inefficient  <cit> , and therefore new resistant cultivars or more efficient antifungal control agents are required.
since the early 1990s, resistance genes  against various pathogens and pests have been isolated from important crop species such as barley, tomato and rice , although most of the r genes known today were isolated from model plant arabidopsis.
these tend to represent qualitative dominant resistance associated with simple mendelian genetics.
in fungal pathosystems, such genes confer resistance to biotrophs and hemi-biotrophs.
resistance to necrotrophic fungal pathogens, by contrast, is often quantitative.
not surprisingly, the genetic basis of resistance is poorly understood and relatively few resistance genes have been characterised.
those that have been reported involve specific interactions with host selective toxins  and are predominantly recessive in nature  presumably by loss or alteration of gene encoding hst targets.
there is evidence to suggest a hst may promote a hypersensitive-like response by acting through a resistance gene leading to cell death  <cit> .
by contrast, of the two necrotrophic resistance genes isolated to date, the race-specific hm <dig> detoxifying gene in maize  <cit>  is dominant and the asc- <dig> longevity assurance gene homolog in tomato  <cit>  is semi-dominant.
the genetic tools to clone and characterize r genes are not available in most legumes, making it difficult to study the molecular basis of resistance to diseases.
medicago truncatula, a close relative of alfalfa, is a model system and possesses a small diploid genome that is currently being sequenced, autogamous genetics and various transformation systems.
the significance of m. truncatula lies in its susceptibility to a range of hemi-biotrophs and necrotrophs and thus is increasingly being used to study resistance to species such as aphanomyces euteiches  <cit> , ascochyta lentis, botrytis cinerea and b. fabae  <cit> , colletotrichum trifolii  <cit> , mycosphaerella pinodes  <cit> , phoma medicaginis  <cit> , and phytophthora medicaginis  <cit> .
sources of resistance to p. medicaginis have been previously identified among different medicago spp.
<cit> , and in the genetically diverse south australian research and development institute  m. truncatula core collection  <cit> .
single-seeded sardi core collection m. truncatula accessions were screened for resistance to three virulent western australian p. medicaginis isolates, and accession sa <dig> was shown to be resistant to p. medicaginis omt <dig>  whereas a <dig> and sa <dig> were highly susceptible  <cit> .
in this study we describe the characterisation of the disease response in these accessions macroscopically and cytologically.
the genetic basis of resistance to p. medicaginis in m. truncatula is determined together with genetic map positions for major loci conferring resistance in two different mapping populations.
RESULTS
macroscopic phenotype of resistant and susceptible accessions
medicago truncatula sardi core collection accessions were previously screened for their response to three p. medicaginis isolates.
accession sa <dig> was resistant to p. medicaginis omt <dig>  whereas a <dig> and sa <dig> were susceptible  <cit> .
when leaves of three-week-old sa <dig> plants were spot inoculated, the fungus was limited to the inoculation site and no chlorosis or necrosis was observed 7– <dig> days post infection .
in susceptible interactions the fungus successfully penetrated host cells and spread beyond the inoculation site, accompanied by a halo of chlorosis ahead of the infection zone.
by 7– <dig> dpi infected leaves were entirely chlorotic or necrotic and pycnidia were apparent .
similar symptoms were observed following spray inoculation: small, microscopic hypersensitive response-like lesions and no chlorosis of leaves occurred in resistant sa <dig> ; in the susceptible interactions disease symptoms were visible to the naked eye at  <dig> dpi.
p. medicaginis colonised the surrounding plant tissue, resulting in macroscopic necrotic spots on both leaves and stems surrounded by spreading chlorosis .
as early as  <dig> dpi p. medicaginis produced pycnidia on susceptible leaves.
microscopic disease symptoms of p. medicaginis omt5
histological staining using trypan blue or dioc <dig> was performed to examine differences in the infection process between resistant and susceptible accessions at the cellular level .
following inoculation, p. medicaginis spores germinated on the leaf surface and successful penetration of host cells was observed as early as  <dig> hours post inoculation .
in both interactions, penetration attempts occurred by three routes; directly through stomata followed by penetration of the underlying mesophyll cells , directly through the epidermal cells , or between epidermal cells .
following penetration, fungal colonisation proceeded rapidly in the susceptible accessions sa <dig> and a <dig>  whereas in the resistant accession sa <dig> hyphal development was limited to individual or a few epidermal cells.
reddish-brown diaminobenzidine-tetrahydrochloride  staining to detect endogenous h2o <dig> production in cells at or around the site of penetration was observed in accession sa <dig> .
however, h2o <dig> was inconsistently associated with penetration attempts and was also evident in susceptible accessions.
autofluorescence was also observed around the site of infection in both the susceptible and resistant interaction, indicating the production of phenolics .
resistance of accession sa <dig> to phoma medicaginis omt <dig> in two different mapping populations
accession sa <dig> was crossed with susceptible accessions a <dig> and sa <dig> to create two f <dig> mapping populations.
f <dig> individuals of both crosses showed p. medicaginis omt <dig> disease symptoms equivalent to the susceptible parents, indicating recessive resistance.
f <dig> individuals were screened for their disease phenotype in both the sa <dig> × a <dig>  and sa <dig> × sa <dig>  populations.
both populations differed significantly from a normal distribution .
the sa <dig> × a <dig> population was not chosen for further genetic studies due to poor fertility, the presence of a reciprocal translocation between chromosomes four and eight in accession a <dig>  <cit> , and a large proportion of aberrant f <dig> individuals .
in the sa <dig> × sa <dig> cross, f <dig> families  were phenotyped to confirm individual f <dig> phenotypes.
when the same f <dig> families were inoculated between different experiments, mean disease scores did not alter significantly showing the phenotyping method was reliable and reproducible.
the proportions of sa <dig> × sa <dig> f <dig> families distributed against the mean disease score  was significantly different from a normal distribution .
the relationship between the families' means and the variance within each family is depicted in figure 3b.
this relationship was evaluated using the fain's test which predicts a pattern of maximum variability in intermediate families whenever at least one locus with major effect is involved  <cit> .
analysis of the f <dig> family disease scores using fain's test revealed that the quadratic term was highly significant , indicating one or a few loci with major effects were involved in resistance to p. medicaginis.
1pollen viability was determined by using alexanders stain as described in kamphuis et al.
<cit> .
2delayed germination indicates development at two weeks was equivalent to  <dig> hrs in a normal seedling.
genetic linkage mapping
genetic maps for the sa <dig> × sa <dig> and sa <dig> × a <dig> mapping populations were created using both gene-based and microsatellite markers.
markers were initially selected to be evenly distributed over each linkage group and were obtained from several published sources  <cit> .
a total of  <dig> markers were characterised for the sa <dig> × sa <dig>  and  <dig> for sa <dig> × a <dig>  populations.
forty-six and  <dig> markers for sa <dig> × sa <dig> and sa <dig> × a <dig> respectively were single nucleotide polymorphisms .
the majority of these snps were converted to cleaved amplified polymorphic sequences .
snps that could not be assayed as caps were genotyped using snapshot  single fluorescent nucleotide extension.
a total 53% of markers were polymorphic between sa <dig> and a <dig> and 35% between sa <dig> and sa <dig>  marker polymorphism data, primer sequences, and references for sa <dig> × sa <dig> and for sa <dig> × a <dig> markers have been provided .
the genetic maps for sa <dig> × a <dig> and sa <dig> × sa <dig> are shown in figures  <dig> and  <dig>  respectively.
as expected, the sa <dig> × sa <dig> map was composed of eight linkage groups, in accordance with the basic number of chromosomes in m. truncatula .
the map spans a total of  <dig>  centimorgans  with an average distance between markers of  <dig>  cm.
very few published markers were polymorphic between sa <dig> and sa <dig> on the lower half of linkage group  <dig>
in contrast to the sa <dig> × sa <dig> map, the sa <dig> × a <dig> map was organized into seven linkage groups, with lg <dig> and lg <dig> forming a single linkage group .
these linkage groups have previously been reported as single linkage groups  <cit> .
the establishment of new linkage relationships between lg <dig> and lg <dig> combined with observations of semisterility in hybrids involving a <dig> provides strong evidence that a <dig> bears a reciprocal translocation, which differentiates it from other m. truncatula accessions.
this reciprocal translocation involves the lower arm of chromosome  <dig> and the lower arm of chromosome  <dig>  <cit> .
all publicly available markers in the middle of lg <dig> were monomorphic and lg <dig> was therefore split into two small linkage groups named lg7a and lg7b.
the sa <dig> × a <dig> genetic map spans a total of  <dig>  cm with an average distance between markers of  <dig>  cm.
the linkage groups of the genetic maps presented here are similar in length to previously published maps  <cit>  with the exception of lg <dig>  in sa <dig> × sa <dig> the distance between dk501r and cyspr <dig> was  <dig>  cm, whereas in sa <dig> × a <dig> this distance was  <dig>  cm.
qtl mapping of spring black stem and leaf spot resistance in m. truncatula
two major quantitative trait loci  for resistance to p. medicaginis omt <dig> were identified, one in each mapping population.
in sa <dig> × a <dig>  the effect of the qtl  was significantly different from zero .
the locus was named resistance to the necrotroph phoma medicaginis one  and was located on lg <dig> , tightly linked to aw <dig>  rnpm <dig> explains  <dig> % of the phenotypic variance in the population's response to infection by p. medicaginis omt <dig>  based on a 1– <dig> scale  <cit> .
in sa <dig> × sa <dig>  the qtl effect  was significantly different from zero .
this locus was named resistance to the necrotroph phoma medicaginis two  and was located on lg <dig> , tightly linked to mtb <dig>  rnpm <dig> explains  <dig> % of the phenotypic variance.
in sa <dig> × sa <dig>  a second qtl, rnpm <dig> was located on linkage group one.
this locus is statistically less significant , has a smaller effect , and explains  <dig> % of the total phenotypic variance.
fine mapping rnpm2
to initiate fine mapping of rnpm <dig> on the long arm of chromosome eight, an additional  <dig> f <dig> individuals  were screened for recombination within a  <dig>  cm interval between markers mtb <dig>  mtb <dig> and mtb <dig> .
f <dig> families from the selected recombinant f <dig> individuals were phenotypically characterised.
as mentioned above, locus rnpm <dig> explains  <dig> % of the total variance in the population's response to the fungal infection when measured in a 1– <dig> scale.
however, recombinant families that showed the sa27063-like  phenotype correlated with homozygous sa <dig> alleles at rnpm <dig> in  <dig> % of instances.
the resistant parent consistently showed little or no visible necrotic symptoms, while disease progression in the susceptible parent was variable and ranged from  <dig>  –  <dig> .
the variance in disease scores was therefore partly a function of the continuum of mean quantitative susceptible disease scores and the sum of disease scores in heterozygous f <dig> families.
recombination break point analysis was therefore used to fine map rnpm <dig>  with phenotyping data from thirty-eight informative f <dig> families in conjunction with further polymorphic markers to resolve recombination break points.
the location of rnpm <dig> was mapped to a  <dig>  cm interval between markers h2_16a6a and h2_21h11d .
discussion and
CONCLUSIONS
the main purpose of this research was to examine differences in disease phenotypes and the genetic basis of resistance to p. medicaginis in m. truncatula.
the resistant phenotype was characterized macroscopically by the presence of small hypersensitive response-like spots, whereas susceptible accessions showed typical stem and leaf lesions, within which pycnidia, or fruiting bodies, formed.
in the resistant accession sa <dig> pycnidia were not apparent, or where present they developed following a delay of 2– <dig> weeks, predominantly on senescing leaves.
microscopically, no differences were observed in penetration attempts between resistant and susceptible accessions, and no obvious changes uniquely associated with resistance were evident with the exception of fungal growth being restricted to one or a few plant cells.
production of h2o <dig> was observed in resistant sa27063-infected leaves but was also detectable in susceptible sa <dig> and a <dig>  h2o <dig> has been reported to play an important role in resistance involving a hypersensitive response  <cit> .
build up of reactive oxygen species  is generally observed following penetration by fungi with a hemibiotrophic or biotrophic lifestyle  <cit>  and subsequently leads to resistance against such fungi, while necrotrophic pathogens may stimulate ros production and subsequent cell death to facilitate subsequent infection  <cit> .
examination of inoculated leaf tissue also showed accumulation of autofluorescent phenolic compounds around the infection site in both resistant and susceptible interactions.
phenolic compounds, such as flavonoids and isoflavonoids, are an important aspect in legume plant defence  <cit> , and there is ample evidence for their accumulation in response to fungal pathogens in medicago species  <cit> .
pilot transcriptional profiling at  <dig> hpi using the mt16koli1plus microarray  <cit>  showed significant changes in induced genes related to oxidative burst, cell wall strengthening, lipid metabolism, and the phenylpropanoid pathway leading to isoflavonoid production .
however, similar levels of induction occurred in both the resistant and susceptible interactions.
cytological similarities reported here are supported in oats, where cell death and induction of defence-related responses were observed in response to the hst victorin in sensitive genotypes , and in barley where a susceptible-specific second ros burst was observed in the later stages of the infection  <cit> .
detailed expression and metabolic profiling may help to identify differences in the hr response and the abundance and composition of defence-related compounds and their role in restricting p. medicaginis colonisation.
the lower proportion of polymorphic makers in the sa <dig> × sa <dig> cross suggested this is a narrower cross than sa <dig> × a <dig>  the total map length for sa <dig> × sa <dig>  was smaller than previously reported genetic maps  <cit>  and in the sa <dig> × a <dig> map.
the genetic distance between markers dk501r and cyspr <dig> on lg <dig> in particular was notably smaller than in the sa <dig> × a <dig> map.
lg <dig> corresponds with chromosome three, the largest chromosome of m. truncatula  <cit> .
one explanation for this phenomenon could be that accessions sa <dig> and sa <dig> are sympatric  <cit> , and may share long stretches of homology in chromosome three, resulting in a lack of detectable recombination.
quantitative trait loci  analysis is often used to identify and characterise loci conferring resistance.
qtl mapping allows the roles of specific r-loci to be described, race-specificity of partial resistance genes can be assessed, and interactions between resistance genes, plant development and the environment to be analysed  <cit> .
qtl analysis for p. medicaginis resistance in the sa <dig> × a <dig> mapping population revealed a qtl on chromosome four  and in the sa <dig> × sa <dig> population a qtl on chromosome eight , both recessive in nature.
as explained in the results, although the rnpm <dig> qtl explained a relatively small amount of the total calculated variance for resistance , sa27063-like or resistant individuals in the f <dig> mapping population correlated with individuals homozygous for sa <dig> alleles at rnpm <dig> in approx.
80% of instances.
among the f <dig> families used in fine mapping and predicted to be resistant by their genotype, this proportion remained constant, allowing rnpm <dig> to be mapped to a  <dig>  cm interval between markers h2_16a6a and h2_21h11d by recombination break point analysis.
the proportion of resistant homozygous sa <dig> rnpm <dig> individuals suggested rnpm <dig> is epistatic to one or several cis- or trans-acting genes or regulatory elements, possibly locus rnpm <dig> on lg <dig>  as the detection and accuracy of minor qtls in segregating populations largely depends on map quality and population size, increasing the initial mapping population size or genome-wide analysis of expressed qtls  <cit>  may reveal other minor qtls and will enable thorough examination of the interaction between rnpm <dig> and rnpm <dig>
we did not proceed with fine mapping rnpm <dig> in the sa <dig> × a <dig> cross, due to poor fertility and a large proportion of aberrant phenotypes among the f <dig> progeny , which may cause biased representation of particular gamete genotypes.
furthermore, accession a <dig> bears a reciprocal translocation, involving chromosomes four and eight, and exhibits reduced pollen viability  <cit> .
all previously published m. truncatula genetic maps used accession a <dig> or a closely related line, j <dig>  as one of the parents.
the sa <dig> × sa <dig> genetic map is the first map produced in m. truncatula not involving a <dig> as a parental line and is therefore useful in identifying the correct position of ambiguously placed markers in a17-derived genetic maps.
the reciprocal translocation involves markers distal to rnpm <dig> at approximately  <dig> cm or less and is therefore unlikely to affect the detection of this qtl among sa <dig> × a <dig> progeny.
the simplest explanation for the existence of two separate qtls among progeny in two crosses involving the same resistant parent is that genotype-specific susceptibility loci may interact with undetermined p. medicaginis omt <dig> virulence factors or hsts.
the rnpm <dig> locus is tightly linked to marker aw <dig> on bac ac <dig>  which is in a contig containing a cluster of tir-nbs-lrr and disease resistance protein-like genes  <cit> , while no rgas are apparent in the genomic sequence of the reference accession a <dig> at the rnpm <dig> locus.
the recessive host genotype-specific nature of rnpm <dig> and rnpm <dig> bears similarities to the race-specific pyrenophora tritici-repentis toxin insensitivity qtls in wheat  <cit> , where multiple, isolate-specific hsts interact with a given host genotype to produce a susceptible phenotype.
to date, no p. medicaginis phytotoxins have been characterised in vivo during infection.
however, the presence of chlorotic symptoms in susceptible m. truncatula accessions inoculated with p. medicaginis in advance of the fungal hyphae, and the large number of toxins produced by other phoma species suggests that susceptible plants with rnpm <dig> and rnpm <dig> are sensitive to a toxin and variation in these genes  results in lack of sensitivity.
future work will be directed at further fine mapping and isolation of rnpm <dig>  in conjunction with gene expression studies to identify genes controlling resistance and candidates for rnpm <dig> in the region of interest.
in addition, the possible involvement of fungal phytotoxins during infection will be characterised and their interaction with the resistance qtl evaluated.

 phoma medicaginis is the causal agent of spring black stem and leaf spot in alfalfa, a major fodder and forage crop in temperate and mediterranean regions. studies in four north-eastern us states have shown foliar necrotrophs, principally p. medicaginis, resulted in estimated average yield losses of over 13%. for harvests where significant yield losses occurred, this figure rose to over 19%. p. medicaginis also causes disease on the model legume medicago truncatula, or barrel medic, which is used in australia in ley rotations to enhance soil nitrogen. in susceptible cultivars, reduction in seed and herbage yields, and almost complete defoliation and premature death has been reported, with infected cultivars exhibiting an average seed weight reduction of %. chemical and cultural control of foliar necrotrophs like p. medicaginis has proved to be expensive and inefficient, and therefore new resistant cultivars or more efficient antifungal control agents are required. since the early 1990s, resistance genes against various pathogens and pests have been isolated from important crop species such as barley, tomato and rice, although most of the r genes known today were isolated from model plant arabidopsis. these tend to represent qualitative dominant resistance associated with simple mendelian genetics. in fungal pathosystems, such genes confer resistance to biotrophs and hemi-biotrophs. resistance to necrotrophic fungal pathogens, by contrast, is often quantitative. not surprisingly, the genetic basis of resistance is poorly understood and relatively few resistance genes have been characterised. those that have been reported involve specific interactions with host selective toxins and are predominantly recessive in nature presumably by loss or alteration of gene encoding hst targets. there is evidence to suggest a hst may promote a hypersensitive-like response by acting through a resistance gene leading to cell death. by contrast, of the two necrotrophic resistance genes isolated to date, the race-specific hm detoxifying gene in maize is dominant and the asc- longevity assurance gene homolog in tomato is semi-dominant. the genetic tools to clone and characterize r genes are not available in most legumes, making it difficult to study the molecular basis of resistance to diseases. medicago truncatula, a close relative of alfalfa, is a model system and possesses a small diploid genome that is currently being sequenced, autogamous genetics and various transformation systems. the significance of m. truncatula lies in its susceptibility to a range of hemi-biotrophs and necrotrophs and thus is increasingly being used to study resistance to species such as aphanomyces euteiches, ascochyta lentis, botrytis cinerea and b. fabae, colletotrichum trifolii, mycosphaerella pinodes, phoma medicaginis, and phytophthora medicaginis. sources of resistance to p. medicaginis have been previously identified among different medicago spp., and in the genetically diverse south australian research and development institute m. truncatula core collection. single-seeded sardi core collection m. truncatula accessions were screened for resistance to three virulent western australian p. medicaginis isolates, and accession sa was shown to be resistant to p. medicaginis omt whereas a and sa were highly susceptible. in this study we describe the characterisation of the disease response in these accessions macroscopically and cytologically. the genetic basis of resistance to p. medicaginis in m. truncatula is determined together with genetic map positions for major loci conferring resistance in two different mapping populations.  macroscopic phenotype of resistant and susceptible accessions medicago truncatula sardi core collection accessions were previously screened for their response to three p. medicaginis isolates. accession sa was resistant to p. medicaginis omt whereas a and sa were susceptible. when leaves of three-week-old sa plants were spot inoculated, the fungus was limited to the inoculation site and no chlorosis or necrosis was observed 7– days post infection. in susceptible interactions the fungus successfully penetrated host cells and spread beyond the inoculation site, accompanied by a halo of chlorosis ahead of the infection zone. by 7– dpi infected leaves were entirely chlorotic or necrotic and pycnidia were apparent. similar symptoms were observed following spray inoculation: small, microscopic hypersensitive response-like lesions and no chlorosis of leaves occurred in resistant sa; in the susceptible interactions disease symptoms were visible to the naked eye at dpi. p. medicaginis colonised the surrounding plant tissue, resulting in macroscopic necrotic spots on both leaves and stems surrounded by spreading chlorosis. as early as dpi p. medicaginis produced pycnidia on susceptible leaves. microscopic disease symptoms of p. medicaginis omt5 histological staining using trypan blue or dioc was performed to examine differences in the infection process between resistant and susceptible accessions at the cellular level. following inoculation, p. medicaginis spores germinated on the leaf surface and successful penetration of host cells was observed as early as hours post inoculation. in both interactions, penetration attempts occurred by three routes; directly through stomata followed by penetration of the underlying mesophyll cells, directly through the epidermal cells, or between epidermal cells. following penetration, fungal colonisation proceeded rapidly in the susceptible accessions sa and a whereas in the resistant accession sa hyphal development was limited to individual or a few epidermal cells. reddish-brown diaminobenzidine-tetrahydrochloride staining to detect endogenous h2o production in cells at or around the site of penetration was observed in accession sa. however, h2o was inconsistently associated with penetration attempts and was also evident in susceptible accessions. autofluorescence was also observed around the site of infection in both the susceptible and resistant interaction, indicating the production of phenolics. resistance of accession sa to phoma medicaginis omt in two different mapping populations accession sa was crossed with susceptible accessions a and sa to create two f mapping populations. f individuals of both crosses showed p. medicaginis omt disease symptoms equivalent to the susceptible parents, indicating recessive resistance. f individuals were screened for their disease phenotype in both the sa × a and sa × sa populations. both populations differed significantly from a normal distribution. the sa × a population was not chosen for further genetic studies due to poor fertility, the presence of a reciprocal translocation between chromosomes four and eight in accession a, and a large proportion of aberrant f individuals. in the sa × sa cross, f families were phenotyped to confirm individual f phenotypes. when the same f families were inoculated between different experiments, mean disease scores did not alter significantly showing the phenotyping method was reliable and reproducible. the proportions of sa × sa f families distributed against the mean disease score was significantly different from a normal distribution. the relationship between the families' means and the variance within each family is depicted in figure 3b. this relationship was evaluated using the fain's test which predicts a pattern of maximum variability in intermediate families whenever at least one locus with major effect is involved. analysis of the f family disease scores using fain's test revealed that the quadratic term was highly significant, indicating one or a few loci with major effects were involved in resistance to p. medicaginis. 1pollen viability was determined by using alexanders stain as described in kamphuis et al.. 2delayed germination indicates development at two weeks was equivalent to hrs in a normal seedling. genetic linkage mapping genetic maps for the sa × sa and sa × a mapping populations were created using both gene-based and microsatellite markers. markers were initially selected to be evenly distributed over each linkage group and were obtained from several published sources. a total of markers were characterised for the sa × sa and for sa × a populations. forty-six and markers for sa × sa and sa × a respectively were single nucleotide polymorphisms. the majority of these snps were converted to cleaved amplified polymorphic sequences. snps that could not be assayed as caps were genotyped using snapshot single fluorescent nucleotide extension. a total 53% of markers were polymorphic between sa and a and 35% between sa and sa marker polymorphism data, primer sequences, and references for sa × sa and for sa × a markers have been provided. the genetic maps for sa × a and sa × sa are shown in figures and respectively. as expected, the sa × sa map was composed of eight linkage groups, in accordance with the basic number of chromosomes in m. truncatula. the map spans a total of centimorgans with an average distance between markers of cm. very few published markers were polymorphic between sa and sa on the lower half of linkage group in contrast to the sa × sa map, the sa × a map was organized into seven linkage groups, with lg and lg forming a single linkage group. these linkage groups have previously been reported as single linkage groups. the establishment of new linkage relationships between lg and lg combined with observations of semisterility in hybrids involving a provides strong evidence that a bears a reciprocal translocation, which differentiates it from other m. truncatula accessions. this reciprocal translocation involves the lower arm of chromosome and the lower arm of chromosome. all publicly available markers in the middle of lg were monomorphic and lg was therefore split into two small linkage groups named lg7a and lg7b. the sa × a genetic map spans a total of cm with an average distance between markers of cm. the linkage groups of the genetic maps presented here are similar in length to previously published maps with the exception of lg in sa × sa the distance between dk501r and cyspr was cm, whereas in sa × a this distance was cm. qtl mapping of spring black stem and leaf spot resistance in m. truncatula two major quantitative trait loci for resistance to p. medicaginis omt were identified, one in each mapping population. in sa × a the effect of the qtl was significantly different from zero. the locus was named resistance to the necrotroph phoma medicaginis one and was located on lg, tightly linked to aw rnpm explains % of the phenotypic variance in the population's response to infection by p. medicaginis omt based on a 1– scale. in sa × sa the qtl effect was significantly different from zero. this locus was named resistance to the necrotroph phoma medicaginis two and was located on lg, tightly linked to mtb rnpm explains % of the phenotypic variance. in sa × sa a second qtl, rnpm was located on linkage group one. this locus is statistically less significant, has a smaller effect, and explains % of the total phenotypic variance. fine mapping rnpm2 to initiate fine mapping of rnpm on the long arm of chromosome eight, an additional f individuals were screened for recombination within a cm interval between markers mtb mtb and mtb. f families from the selected recombinant f individuals were phenotypically characterised. as mentioned above, locus rnpm explains % of the total variance in the population's response to the fungal infection when measured in a 1– scale. however, recombinant families that showed the sa27063-like phenotype correlated with homozygous sa alleles at rnpm in % of instances. the resistant parent consistently showed little or no visible necrotic symptoms, while disease progression in the susceptible parent was variable and ranged from –. the variance in disease scores was therefore partly a function of the continuum of mean quantitative susceptible disease scores and the sum of disease scores in heterozygous f families. recombination break point analysis was therefore used to fine map rnpm with phenotyping data from thirty-eight informative f families in conjunction with further polymorphic markers to resolve recombination break points. the location of rnpm was mapped to a cm interval between markers h2_16a6a and h2_21h11d. discussion and  the main purpose of this research was to examine differences in disease phenotypes and the genetic basis of resistance to p. medicaginis in m. truncatula. the resistant phenotype was characterized macroscopically by the presence of small hypersensitive response-like spots, whereas susceptible accessions showed typical stem and leaf lesions, within which pycnidia, or fruiting bodies, formed. in the resistant accession sa pycnidia were not apparent, or where present they developed following a delay of 2– weeks, predominantly on senescing leaves. microscopically, no differences were observed in penetration attempts between resistant and susceptible accessions, and no obvious changes uniquely associated with resistance were evident with the exception of fungal growth being restricted to one or a few plant cells. production of h2o was observed in resistant sa27063-infected leaves but was also detectable in susceptible sa and a h2o has been reported to play an important role in resistance involving a hypersensitive response. build up of reactive oxygen species is generally observed following penetration by fungi with a hemibiotrophic or biotrophic lifestyle and subsequently leads to resistance against such fungi, while necrotrophic pathogens may stimulate ros production and subsequent cell death to facilitate subsequent infection. examination of inoculated leaf tissue also showed accumulation of autofluorescent phenolic compounds around the infection site in both resistant and susceptible interactions. phenolic compounds, such as flavonoids and isoflavonoids, are an important aspect in legume plant defence, and there is ample evidence for their accumulation in response to fungal pathogens in medicago species. pilot transcriptional profiling at hpi using the mt16koli1plus microarray showed significant changes in induced genes related to oxidative burst, cell wall strengthening, lipid metabolism, and the phenylpropanoid pathway leading to isoflavonoid production. however, similar levels of induction occurred in both the resistant and susceptible interactions. cytological similarities reported here are supported in oats, where cell death and induction of defence-related responses were observed in response to the hst victorin in sensitive genotypes, and in barley where a susceptible-specific second ros burst was observed in the later stages of the infection. detailed expression and metabolic profiling may help to identify differences in the hr response and the abundance and composition of defence-related compounds and their role in restricting p. medicaginis colonisation. the lower proportion of polymorphic makers in the sa × sa cross suggested this is a narrower cross than sa × a the total map length for sa × sa was smaller than previously reported genetic maps and in the sa × a map. the genetic distance between markers dk501r and cyspr on lg in particular was notably smaller than in the sa × a map. lg corresponds with chromosome three, the largest chromosome of m. truncatula. one explanation for this phenomenon could be that accessions sa and sa are sympatric, and may share long stretches of homology in chromosome three, resulting in a lack of detectable recombination. quantitative trait loci analysis is often used to identify and characterise loci conferring resistance. qtl mapping allows the roles of specific r-loci to be described, race-specificity of partial resistance genes can be assessed, and interactions between resistance genes, plant development and the environment to be analysed. qtl analysis for p. medicaginis resistance in the sa × a mapping population revealed a qtl on chromosome four and in the sa × sa population a qtl on chromosome eight, both recessive in nature. as explained in the results, although the rnpm qtl explained a relatively small amount of the total calculated variance for resistance, sa27063-like or resistant individuals in the f mapping population correlated with individuals homozygous for sa alleles at rnpm in approx. 80% of instances. among the f families used in fine mapping and predicted to be resistant by their genotype, this proportion remained constant, allowing rnpm to be mapped to a cm interval between markers h2_16a6a and h2_21h11d by recombination break point analysis. the proportion of resistant homozygous sa rnpm individuals suggested rnpm is epistatic to one or several cis- or trans-acting genes or regulatory elements, possibly locus rnpm on lg as the detection and accuracy of minor qtls in segregating populations largely depends on map quality and population size, increasing the initial mapping population size or genome-wide analysis of expressed qtls may reveal other minor qtls and will enable thorough examination of the interaction between rnpm and rnpm we did not proceed with fine mapping rnpm in the sa × a cross, due to poor fertility and a large proportion of aberrant phenotypes among the f progeny, which may cause biased representation of particular gamete genotypes. furthermore, accession a bears a reciprocal translocation, involving chromosomes four and eight, and exhibits reduced pollen viability. all previously published m. truncatula genetic maps used accession a or a closely related line, j as one of the parents. the sa × sa genetic map is the first map produced in m. truncatula not involving a as a parental line and is therefore useful in identifying the correct position of ambiguously placed markers in a17-derived genetic maps. the reciprocal translocation involves markers distal to rnpm at approximately cm or less and is therefore unlikely to affect the detection of this qtl among sa × a progeny. the simplest explanation for the existence of two separate qtls among progeny in two crosses involving the same resistant parent is that genotype-specific susceptibility loci may interact with undetermined p. medicaginis omt virulence factors or hsts. the rnpm locus is tightly linked to marker aw on bac ac which is in a contig containing a cluster of tir-nbs-lrr and disease resistance protein-like genes, while no rgas are apparent in the genomic sequence of the reference accession a at the rnpm locus. the recessive host genotype-specific nature of rnpm and rnpm bears similarities to the race-specific pyrenophora tritici-repentis toxin insensitivity qtls in wheat, where multiple, isolate-specific hsts interact with a given host genotype to produce a susceptible phenotype. to date, no p. medicaginis phytotoxins have been characterised in vivo during infection. however, the presence of chlorotic symptoms in susceptible m. truncatula accessions inoculated with p. medicaginis in advance of the fungal hyphae, and the large number of toxins produced by other phoma species suggests that susceptible plants with rnpm and rnpm are sensitive to a toxin and variation in these genes results in lack of sensitivity. future work will be directed at further fine mapping and isolation of rnpm in conjunction with gene expression studies to identify genes controlling resistance and candidates for rnpm in the region of interest. in addition, the possible involvement of fungal phytotoxins during infection will be characterised and their interaction with the resistance qtl evaluated. 
knowledge of the genetic basis of plant resistance to necrotrophic pathogens is incomplete and has been characterised in relatively few pathosystems. both loci are recessive in nature, and the simplest explanation for the existence of two separate qtls is the occurrence of host genotype-specific susceptibility loci that may interact with undetermined p. medicaginis virulence factors. in this study, the cytology and genetics of resistance to spring black stem and leaf spot caused by phoma medicaginis, an economically important necrotrophic pathogen of medicago spp., was examined in the model legume m. truncatula. two f populations segregating for resistance to spring black stem and leaf spot were established between sa and the two susceptible accessions, a and sa the cross between sa and a represented a wider cross than between sa and sa as evidenced by higher genetic polymorphism, reduced fertility and aberrant phenotypes of f progeny. in the sa × a f population a highly significant quantitative trait locus named resistance to the necrotroph phoma medicaginis one genetically mapped to the top arm of linkage group. rnpm explained % of the phenotypic variance in the population's response to infection depicted on a 1– scale and was tightly linked to marker aw a second highly significant qtl, rnpm was located on the lower arm of lg in the sa × sa map. rnpm explained % of the phenotypic variance and was fine mapped to a cm interval between markers h2_16a6a and h2_21h11d. no unique cytological differences were observed during early infection between the resistant and susceptible genotypes, except pathogen growth was restricted to one or a few host cells in sa in both interactions reactive oxygen intermediates and phenolic compounds were produced, and cell death occurred. rnpm is tightly linked to a cluster of toll/interleukin receptor-nucleotide binding site-leucine-rich repeat genes and disease resistance protein-like genes, while no resistance gene analogues are apparent in the genomic sequence of the reference accession a at the rnpm locus. the induction of defence responses and cell death in the susceptible interaction following infection by p. medicaginis suggested this pathogen is not negatively affected by these responses and may promote them. a qtl for resistance was revealed in each of two populations derived from crosses between a resistant accession and two different susceptible accessions.
 knowledge of the genetic basis of plant resistance to necrotrophic pathogens is incomplete and has been characterised in relatively few pathosystems. in this study, the cytology and genetics of resistance to spring black stem and leaf spot caused by phoma medicaginis, an economically important necrotrophic pathogen of medicago spp., was examined in the model legume m. truncatula.  macroscopically, the resistant response of accession sa was characterised by small, hypersensitive-like spots following inoculation while the susceptible interaction with accessions a and sa showed necrotic lesions and spreading chlorosis. no unique cytological differences were observed during early infection between the resistant and susceptible genotypes, except pathogen growth was restricted to one or a few host cells in sa in both interactions reactive oxygen intermediates and phenolic compounds were produced, and cell death occurred. two f populations segregating for resistance to spring black stem and leaf spot were established between sa and the two susceptible accessions, a and sa the cross between sa and a represented a wider cross than between sa and sa as evidenced by higher genetic polymorphism, reduced fertility and aberrant phenotypes of f progeny. in the sa × a f population a highly significant quantitative trait locus named resistance to the necrotroph phoma medicaginis one genetically mapped to the top arm of linkage group. rnpm explained % of the phenotypic variance in the population's response to infection depicted on a 1– scale and was tightly linked to marker aw a second highly significant qtl, rnpm was located on the lower arm of lg in the sa × sa map. rnpm explained % of the phenotypic variance and was fine mapped to a cm interval between markers h2_16a6a and h2_21h11d. rnpm is tightly linked to a cluster of toll/interleukin receptor-nucleotide binding site-leucine-rich repeat genes and disease resistance protein-like genes, while no resistance gene analogues are apparent in the genomic sequence of the reference accession a at the rnpm locus.  the induction of defence responses and cell death in the susceptible interaction following infection by p. medicaginis suggested this pathogen is not negatively affected by these responses and may promote them. a qtl for resistance was revealed in each of two populations derived from crosses between a resistant accession and two different susceptible accessions. both loci are recessive in nature, and the simplest explanation for the existence of two separate qtls is the occurrence of host genotype-specific susceptibility loci that may interact with undetermined p. medicaginis virulence factors. 