BACKGROUND
agriculture is facing enormous challenges to feed a growing population in the face of rapidly evolving pests and pathogens.
a critical component for addressing these challenges is to breed for increased disease resistance in crop species to avoid the need for costly and potentially environmentally damaging pesticides.
the major cereal crops feed over two thirds of the world’s population and yet the production of these crops continues to be challenged by pests and diseases with at least 30% of global food production lost to pathogens  <cit> .
sorghum  moench) is a c <dig> cereal grain crop that provides staple food for over  <dig> million people in the semi-arid tropics of africa and asia, in addition to being an important source of feed for livestock.
amongst the cereals, sorghum is one of the best adapted to drought and high temperatures, and will play an increasingly important role in meeting the challenges of feeding the world’s growing population.
in recent times, sorghum has become an attractive feedstock alternative for use in the production of bio-ethanol fuel.
however, its productivity is often jeopardised by foliar fungal diseases.
among the fungal diseases, leaf rust causes significant crop damage in sorghum  <cit> , with rust diseases being among the most widespread and economically important diseases of cereals world-wide, e.g.
<cit> .
sorghum rust, caused by puccinia purpurea cooke, is widely distributed and occurs in almost all sorghum growing areas of the world  <cit> .
leaf rust frequently predisposes sorghum to other major diseases and agronomic problems, such as the fusarium stalk rots, charcoal rot, grain mould, anthracnose and lodging  <cit> .
in addition to indirect yield losses through predisposition to other diseases, sorghum leaf rust can cause direct yield losses of up to 50% depending on favourable environmental conditions for disease development and cultivar susceptibility  <cit> .
in australia, leaf rust has the most significant impact on grain yield of all fungal leaf diseases, causing up to 13% yield losses  <cit> .
the disease is seen every year on most sorghum crops, usually after flowering, when the leaves may be covered by the uredinia and telia of the fungus  <cit> .
due to the influence of environmental and climatic conditions on the development of the disease and the cost of fungicides, genetic resistance offers the best long-term solution for the management of leaf rust in sorghum.
as such, better understanding of the genetic control of leaf rust resistance would provide sorghum breeders with critical knowledge to develop new resistant varieties.
the genetic architecture of complex traits is frequently controlled by multiple genes or alleles that vary with genetic background  <cit> .
a single mapping population study can therefore explain only a small part of the global genetic architecture of a complex trait and limits the identification of potential genomic regions due to the absence or monomorphic presence of alleles contributing to the genetic control of a complex trait such as leaf rust resistance  <cit> .
initial studies into the inheritance of leaf rust resistance in sorghum identified a single dominant gene, pu, conferring rust resistance in sweet sorghum crosses  <cit> .
however, subsequent studies identified rust resistance to be polygenic in nature, with multiple genes and genomic regions identified  <cit> .
therefore, the comparison of qtl detected in multiple populations and multiple environments is particularly important for dissecting the genetic control of polygenic disease resistance and permits investigation of the degree to which the underlying genes contribute to variation in the phenotype under different genetic backgrounds and environmental conditions  <cit> .
furthermore, traits associated with the physiological development and age of the plant have been implicated in indirectly impacting disease response, with previous studies demonstrating that growth stage can impact on the degree of disease susceptibility in a range of crops e.g.
<cit> .
therefore, a multi-trait analysis approach enables the investigation of potentially pleiotropic disease response qtl.
in this study, a combination of conventional qtl analysis and genome wide association analysis  was used to determine the genetic architecture of leaf rust resistance in sorghum using three bi-parental mapping populations and a set of elite breeding lines phenotyped in hybrid combination with multiple testers.
all populations were phenotyped not only for rust infection response, but also for height and maturity.
further, the availability of the sorghum whole genome sequence  <cit>  and genetic linkage map-based resources, e.g.
<cit> , provided opportunities to compare qtl for rust resistance identified in the current study with previously reported qtl in both sorghum and maize, in addition to previously reported gene families associated with disease resistance.
RESULTS
phenotypic data variability
the predicted means, ranges, and standard deviations for the traits measured for the progeny of the two ril populations are detailed in table  <dig>  rust infection responses of the five parents in the three bi-parental populations are presented in additional file 1: figure s <dig>  heritability for rust resistance was high in all three bi-parental populations .
in the s <dig> population, the female parent  had higher levels of rust resistance than the male parent ;  <dig>  versus  <dig>  in the s <dig> population, the male parent  exhibited partial resistance to rust , in contrast to the high level of susceptibility  exhibited by the female parent .
for s <dig>  the rust resistant parent icsv <dig> was much later maturing  and much taller  than r <dig>  between trait correlation was low .
for s <dig>  r931945-2- <dig> was slightly later maturing and shorter than is <dig>  the rils showed transgressive segregation for all three traits.
the predicted means, ranges, and standard deviations for all three traits scored in the s <dig> population are detailed in table  <dig>  the s. bicolor subsp.
verticilliflorum parent could not be grown in field trials because of its weedy nature.
the predicted means, ranges, and standard deviations for the ayt association mapping set across the  <dig> male testers and  <dig> location combinations are detailed in additional file 2: table s <dig>  heritability for rust resistance was found to be higher at the liverpool plains site , in comparison to the dalby site .
the relative rust pressure was found to be reduced at the dalby site, in comparison to the other trials included in the study, through the comparison of rust response scores of check genotypes .
the trials were not highly correlated ; genotype × environment interaction was observed using a second order factor analytic  site structure where only 68% of the variation was accounted for by the first factor , and additionally correlations across sites and within each male tester genotypes were higher than on a per site basis .table  <dig>
predicted mean  values of rust infection score, height and maturity for the two ril populations  and parental lines in the hrs <dig> trial, plus the standard deviation  and range  for each population
population
trait
x¯parenta
x¯parentb
x¯progeny
σ
progeny
min
progeny
max
progeny
1
f
4
population  across multiple trials, plus the standard deviation  and range
trial
trait
x¯progeny
σ
progeny
min
progeny
max
progeny
bil2003irr
bil2003nonirr
bil2003overall
bil2003irr
bil2003nonirr
bil2003overall
bil2003irr
bil2003nonirr
bil2003overall
qtl analysis
the results of the qtl analysis for each trait in each population are shown in tables  <dig>   <dig>  and  <dig> .table  <dig>
summary of rust resistance , maturity  and height  qtl identified in the s <dig> population, detailing the qtl position, 2-lod confidence interval , flanking markers, peak lod value, total trait variance explained , additive effect, and significance level
qtl id
lg
peak cm
a
ci
flanking markers
lod
r
2
b
additive
c
sig
d
apeak position in cm based on the s <dig> genetic linkage map; bthe amount of total trait variance explained by a qtl at this locus, as %; cthe allelic effects are calculated as the effect of substitution of aa  allele by bb  allele; d*suggestive ; **significant ; ***highly significant .
summary of rust resistance , maturity  and height  qtl identified in the s <dig> population, detailing the qtl position, 2-lod confidence interval , flanking markers, peak lod value, total trait variance explained , additive effect, and significance level
qtl id
lg
peak
a
ci
flanking markers
lod
r
2b
additive
c
sig
d
apeak position in cm based on the s <dig> genetic linkage map; bthe amount of total trait variance explained by a qtl at this locus, as %; cthe allelic effects are calculated as the effect of substitution of aa  allele by bb  allele; d*suggestive ; **significant ; ***highly significant .
summary of rust resistance qtl identified in the s <dig> population detailing the qtl location on consensus map, additive effect and significance level across  <dig> sites
allele effects
b
qtl id
lg
peak cm
a
bil03
irr
bil03
nonirr
bil03
overall
apeak position with maximum –log10p; bthe allelic effects are calculated as the effect of substitution of aa  allele by bb  allele.
ns: not significant; **significant ; ***highly significant .
rust resistance
in population s <dig>  cim identified two highly significant rust resistance qtl, one on sbi- <dig> and one on sbi-08-ii .
in addition, three suggestive qtl were detected on sbi- <dig>  sbi-06-i and sbi-08-i.
individual qtl explained between  <dig>  to  <dig> % of phenotypic variation in response to rust, with a cumulative total of  <dig> %.
the majority of qtl  had positive allelic effects indicating that the icsv <dig> qtl alleles predominately contributed to an increase in rust resistance.
in population s <dig>  a total of  <dig> qtl for rust resistance were identified located on  <dig> chromosomes.
one highly significant and three significant qtl were identified by cim analysis on sbi- <dig>  sbi- <dig>  sbi- <dig> and sbi- <dig> .
five suggestive qtl were identified on sbi- <dig>  sbi- <dig>  sbi- <dig> and sbi- <dig>  individual qtl explained between  <dig>  to  <dig> % of phenotypic variation, with a cumulative total of  <dig> %.
six of the  <dig> rust resistance qtl had negative effects, indicating that parent r931945-2- <dig> qtl alleles predominately contributed to an increase in rust resistance.
in population s <dig>  fifteen genomic regions were detected with significant marker trait associations  on eight chromosomes .
a further four genomic regions were detected with highly significant marker trait associations .
the majority  of the identified qtl had negative allele effects indicating that the s. bicolor subsp.
verticilliflorum qtl alleles predominantly contributed to an increase in rust resistance.
in the ayt association mapping set,  <dig> genomic regions were identified with suggestive marker trait associations in at least one of the  <dig> tester/location combinations , with over half  identified as significant in two or more of the tester/location combinations.
to combine the results of the association mapping analyses across the six male tester and location combinations, the number of tester/location combinations with a significant marker trait association was calculated for each marker  and plotted against the sorghum consensus map based on a sliding window of 2 cm with a step size of  <dig>  cm .
of the  <dig> qtl identified,  <dig> were identified in a single tester/location combination only and hence can be considered as suggestive qtl regions.
just over 10%  of the qtl were influenced by the genetic background, being identified only with specific male testers across both locations .
a further  <dig> qtl  were location-specific, being identified only in one location, however these included the  <dig> suggestive qtl only identified in a single tester/location combination.
three qtl were identified in all three male tester combinations at a single site only .
a further three qtl were identified in both sites and across all male tester combinations .
the total number of qtl identified with the male tester r986087-2-4- <dig>  across both locations, was almost 50% higher than with either of the other two male testers, r <dig> and r995248;  <dig> qtl vs  <dig> qtl vs  <dig> qtl respectively.
the male line, r986087-2-4- <dig> was produced from a cross between r931945-2- <dig> and sc170-6-17; r931945-2- <dig> being a parental line of s <dig> and s <dig> populations.
of the  <dig> rust resistance qtl identified in both s <dig> and s <dig>   <dig> were derived from r931945-2- <dig>  the graphical genotype of r986087-2-4- <dig> based on previously generated gbs data  indicated that r986087-2-4- <dig> was identical by descent  to r931945-2- <dig> in  <dig> of these  <dig> qtl regions.
in all  <dig> of these regions, a qtl was also identified in the ayt populations with the r986087-2-4- <dig> tester.figure  <dig>
rust resistance qtl from the current study projected onto the sorghum consensus genetic linkage map in comparison with rust qtl identified in previous studies.
the bars to the right of each chromosome indicate rust resistance qtl identified, colour-coded as follows: red: s <dig> ; dark green: s <dig> ; dark blue: s <dig> ; green:  <cit> ; orange;  <cit> ; pink:  <cit> .
the locations of the qtl identified in the ayt association mapping set are represented by a separate heat map, for each lg, detailing qtl density .
maturity
in the s <dig> population, five suggestive qtl for maturity were identified located on four chromosomes , individually explaining between  <dig>  to  <dig> % of the phenotypic variation, with a cumulative total of  <dig> % .
the maturity qtl on sbi- <dig> co-located with the highly significant rust resistance qtl  in the same population , with the r <dig> alleles contributing towards lateness in maturity and rust resistance.
in the s <dig> population, two highly significant qtl on sbi- <dig> and sbi- <dig> were identified, with a cumulative total of  <dig> % of the phenotypic variation explained .
three additional suggestive qtl were identified on sbi- <dig>  sbi- <dig> and sbi- <dig>  individually explaining between  <dig>  to  <dig> % of the phenotypic variation.
none of the maturity qtl co-located with rust qtl in the same population.
both positive and negative additive effects were observed, indicating that the r931945-2- <dig> parent had alleles contributing to both an increase and a decrease in maturity.
in s <dig>   <dig> highly significant qtl were located on eight chromosomes .
fourteen of the  <dig> qtl had positive allele effects indicating that the s. bicolor subsp.
verticilliflorum qtl alleles predominantly contributed to an increase in late maturity.
a further two significant qtl regions were identified, both of which had positive allele effects.
overall, approximately a quarter  of the maturity qtl, four highly significant and one significant, in s <dig> co-located with rust qtl in the same population .
the direction of allelic effects was consistent across co-locating qtl, with the rust resistance qtl alleles from s. bicolor subsp.
verticilliflorum always occurring with late maturing alleles from s. bicolor subsp.
verticilliflorum.
in the ayt association mapping set,  <dig> qtl for maturity were identified, eight of which co-located with rust qtl .
height
in the s <dig> population, one significant qtl on sbi- <dig> and three suggestive qtl on sbi- <dig>  sbi- <dig> and sbi- <dig> were identified, with a cumulative total of  <dig> % of phenotypic variation explained .
three of the four qtl had negative allele effects, indicating that the qtl alleles from r <dig> predominantly contributed to a decrease in height.
the height qtl on sbi- <dig> co-located with a suggestive rust qtl on sbi- <dig>  in the same population , and the direction of qtl allele effects were consistent .
in the s <dig> population, three highly significant qtl for height were identified, with a cumulative total of  <dig> % of the phenotypic variation explained, on chromosomes sbi- <dig>  sbi- <dig> and sbi- <dig> .
three further suggestive qtl were identified on sbi- <dig>  sbi- <dig> and sbi- <dig>  the suggestive qtl for height on sbi- <dig>  co-located with a rust qtl  in the same population , while the highly significant height qtl on sbi- <dig> co-located with a maturity qtl.
in both cases the direction of qtl allele effects was consistent with the observations from the s <dig> population .
in s <dig>  only one highly significant qtl for height was identified on sbi- <dig>  while a further  <dig> significant qtl were identified on sbi- <dig>  sbi- <dig>  sbi- <dig> and sbi- <dig> .
five of the height qtl identified had positive allele effects indicating that the s. bicolor subsp.
verticilliflorum qtl alleles predominantly contributed to an increase in height.
the significant qtl for height on sbi- <dig>  co-located with qtl for both rust and maturity, while the significant qtl for height on sbi- <dig> co-located within 10 cm a rust qtl  .
as previously, the direction of allele effects of the co-locating qtl was consistent with observations from the s <dig> and s <dig> populations .
in the ayt association mapping set,  <dig> qtl for height was identified on sbi- <dig>  which co-located with rust and maturity qtl also identified in the ayt population .
stay-green
due to its association with carbon stress, the stay-green trait was also analysed in the ayt association mapping set.
it was visually scored as a leaf senescence rating from 1– <dig>  <cit> , across the same three male tester genotypes and two sites, with one trial site in common with the previous analyses, and analysed using the same methodology as described for the other traits, identifying nine significant qtl .
qtl projection on consensus map
all the qtl identified in each of the three bi-parental populations and the ayt association mapping set were projected on the consensus map for all three traits  and compared to rust, maturity and height qtl identified previously.
across the four populations in this study,  <dig> significant or highly significant qtl and  <dig> suggestive qtl for rust resistance were identified and projected onto the consensus map, in addition to the  <dig> previously reported qtl for rust resistance  <cit> , representing a total of  <dig> unique regions .
rust qtl were identified on all chromosomes, however the genome-wide distribution was uneven, with only  <dig> unique qtl identified on sbi- <dig> in contrast to  <dig> rust qtl located on sbi- <dig>  the syntenic locations of  <dig> rust qtl identified in maize  <cit>  were also projected onto the consensus map and compared to the locations of the rust qtl from sorghum .
ten of the previously reported sorghum rust qtl co-located with rust qtl identified in the current study; one genomic region described by  <cit>  was found to be unique among the sorghum rust studies investigated, however this qtl co-located with maize rust qtl  <cit> .
in total, of the  <dig> rust resistance qtl identified in maize, over 80%  co-located with rust qtl identified in sorghum  .
the  <dig> unique rust qtl regions identified were further categorised according to their co-location with height and maturity qtl, to distinguish between 1) qtl unique to rust within a population and 2) multi-trait effect qtl, with rust qtl co-locating with qtl for other traits within a population .
of the  <dig> unique genomic regions,  <dig> were categorised as being unique to rust, whereas  <dig> were not unique to rust, and co-located with either height, maturity or stay-green qtl identified in the same population.
the correspondence of the two different types of rust resistance qtl locations and the nbs-lrr gene family members, previously reported to be associated with disease resistance  <cit> , were investigated.
the nbs-encoding genes were significantly enriched in the regions of the genome containing unique rust disease resistance qtl , in contrast to the rust qtl that co-located with other traits .
in particular, the major nbs-encoding rust resistance gene rph1- <dig>  homologous with the maize rp1-d gene, previously described in sorghum by  <cit>  and also believed to be the pu gene  <cit> , co-located with qtl from the s <dig> population  and the ayt association mapping set  .
additionally, the wheat rust resistance gene, lr <dig>  previously described by  <cit> , co-located with qtl from the s <dig> population  and the ayt association mapping set  , in addition to rust qtl identified in maize  <cit> .
both parental genotypes of the s <dig> and s <dig> populations were included in the recently described sorghum resequencing data set  <cit> , enabling sequence analysis of underlying candidate genes.
in silico analysis for missense mutations revealed a polymorphism leading to an amino-acid change that departed from the peptide arising from the predicted gene models  <cit>  in the candidate gene for lr <dig>  which co-located with a rust resistance qtl in the s <dig> population .
the thirty-eight qtl for maturity identified in s <dig>  s <dig>  s <dig> and the ayt association mapping set were projected onto the consensus map and compared to  <dig> maturity qtl reported previously.
of the  <dig> maturity qtl identified in the current study,  <dig> co-located with maturity qtl described previously .
of the remaining  <dig> qtl,  <dig> co-located across the populations included in the current study, with only  <dig> novel maturity qtl identified .
two major-effect maturity loci previously projected onto the sorghum consensus map  <cit>  were identified in the current study; ma <dig> was identified in s <dig> , and ma <dig> was identified in populations s <dig> and s <dig> .
similarly, the seventeen qtl for height identified in s <dig>  s <dig>  s <dig> and the ayt association mapping set were projected onto the consensus map and compared to  <dig> height qtl reported previously.
sixteen of the  <dig> height qtl identified co-located with height qtl previously described , including the  <dig> major effect height loci previously projected onto the consensus map; dw <dig> on sbi- <dig> , dw <dig> on sbi- <dig>  and dw <dig> on sbi- <dig> .
discussion
the rusts are a major pathogen of cereal crops with the potential to cause large reductions in yield and have as a result been the focus of ongoing plant breeding effort particularly in the winter cereals.
the challenge for breeders in these species has been to produce stable resistance due to the rapidly evolving nature of the pathogen.
sorghum is a major summer cereal crop that is also a host for a rust pathogen puccinia purpurea.
leaf rust occurs in almost all sorghum growing areas of the world  <cit>  and can cause significant yield losses in the crop.
in australia, rust infection occurs in most sorghum crops with the severity of epidemics being greater in humid environments and in late-sown crops  <cit> .
despite the prevalence of the disease in sorghum, it is not subject to the large damaging rust epidemics which regularly occur in winter cereals in australia.
understanding the genetic basis of rust in sorghum will be important for improving the crop itself as well as providing a comparison and potential insight into the architecture of the trait in other species.
this study used natural field infection data from multiple populations grown in multiple environments using a combination of conventional linkage mapping and association mapping to investigate the genetic control of rust resistance and the potential for multi-trait effect qtl.
rust resistance is a polygenic trait controlled by multiple qtl of small effect
the complex, polygenic nature of this trait was confirmed with  <dig> significant or highly significant qtl identified representing over  <dig> unique genomic regions, in addition to  <dig> suggestive qtl representing a further  <dig> unique genomic regions.
comparisons with previous rust resistance qtl studies revealed that  <dig> of the  <dig> previously reported rust resistance qtl in sorghum were also identified in the current study.
the genetic architecture of the trait was largely found to involve multiple qtl of small effects, rather than major genes of large effect, with the largest allele effect reported in the s <dig> population , representing  <dig> % of the total phenotypic variation, and with an average qtl allele effect across all qtl identified of just  <dig> .
this contrasts to the major effect leaf rust qtl commonly reported in other cereals, e.g.
the major effect adult plant resistance gene rph <dig> in barley, where the additive allele effect sizes accounted for 64-85% of the phenotypic variation in adult plants across four field environments  <cit> ; the major effect, “slow-rusting” resistance gene lr <dig> conferring durable adult plant resistance in wheat, explaining up to 55% of the phenotypic variation  <cit> .
this difference in the genetic control of leaf rust resistance across cereals could be partly due to the fact that sorghum is primarily a perennial species in comparison to the strongly annual life-history of the other major cereals, where the need for more stable resistance due to increased plant longevity could favour selection for multiple resistance genes of small effect.
this study encompassed populations with a degree of shared ancestry, which enabled the evaluation of resistance alleles that were identical by descent  in different populations and experiments.
the elite line r931945-2- <dig> was not only a common parent in the s <dig> and s <dig> populations but also a parent of the common male tester r986087-2-4- <dig> used to produce  <dig> of the f <dig> hybrids used in the association mapping set.
qtl alleles from r931945-2- <dig> contributed to increased rust resistance in just under half  of the qtl identified from the two bi-parental populations; of these qtl, seven corresponded to genomic regions that were ibd between r986087-2-4- <dig> and r931945-2- <dig>  in all  <dig> of these regions, a qtl was identified in this location in the ayt association mapping set, in hybrids with r986087-2-4- <dig> as the common tester.
this result suggests that the inheritance of these seven resistance qtl is not completely dominant, but instead either additive or recessive.
this conclusion is also supported by the detection of the vast majority  of the significant qtl in the ayt association mapping set identified across multiple male tester hybrid combinations.
in commercial agricultural settings sorghum cultivars are almost exclusively f <dig> hybrids and as result knowledge of the degree of dominance in traits is of critical importance for their deployment as cultivars.
the ibd analysis also enabled us to determine that two of the r931945-2- <dig> derived rust resistance qtl were identical to rust resistance qtl contributed by ql <dig> in the mapping population used by  <cit> .the stability of these qtl suggests that there has been limited variation in the rust pathotypes over the last 15 years.
in addition the fact that the majority of the rust qtl  was detected at both ayt trial locations suggests that there is relatively low spatial and temporal variation in rust populations.
rust resistance is strongly influenced by the physiological state of the plant
in sorghum, rust infection generally develops later in the crop’s cycle as the plant begins filling grain  <cit> .
this is consistent with the observation of  <cit>  who demonstrated that the growth stage, or physiological age, of groundnut  can impact on the degree of susceptibility to rust infection.
typically plants in vegetative stages are less susceptible to many diseases than those infected at reproductive phases, when the plant is remobilizing carbohydrates during grain-filling  <cit> .
this phenomenon has important implications for assessing rust resistance in plants that vary in maturity or other characters such as height or stay-green which influence the developmental status of the plant and hence the nature and degree of carbohydrate remobilization.
this is particularly important in field grown plants subject to natural infection.
although the association between height and rust response could be related to a variation in microclimate, the association is consistent with the change in source/sink dynamics where earlier maturing plants become vulnerable to infection before later plants and taller plants have greater reserves for remobilization.
carbon stress also plays an important role in drought responsiveness.
plants with the stay-green characteristics have increased functional carbohydrate resources for remobilisation during grain filling  <cit> .
we observed low but consistent, negative phenotypic correlations between the severity of rust infection and both maturity and height.
in all populations and trials early maturity and short stature was associated with rust susceptibility.
this result is consistent with the hypothesis that variation in plant carbohydrate status influences susceptibility to rust.
at the qtl level we found a highly significant coincidence of rust qtl with qtl for height and maturity in the four populations used.
a statistically significant proportion  of the rust qtl co-located with one of these traits in the same population.
a recent synthesis of disease resistance qtl studies in maize found comparable significant associations between disease resistance qtl and maturity qtl in the maize genome  <cit> .
co-locating qtl regions could contain multiple tightly linked genes for resistance and other traits or single genes with a pleiotropic effect across multiple traits.
however the evidence for pleiotropic qtl action in the current study is very strong given the consistency in the direction of qtl allele effects, i.e.
taller and later maturing plants carried the rust resistant qtl allele in the majority of cases .
this is consistent with a recent study analysing the genetic control of rust in sorghum  <cit>  who found rust severity in field grown sorghum was negatively correlated with plant height and maturity.
interestingly, when the same genotypes were artificially infected at a single time point in the glasshouse these correlations were not apparent  <cit> .
this provides further evidence that the physiological state influences either the timing of infection or the rate at which infection develops.
the co-location of 100% of the leaf senescence, stay-green characteristic, qtl identified in the ayt association mapping set with rust qtl, in addition to the consistency of the direct of the effects , provides further evidence to support the role of carbon stress in disease resistance.
additionally, when all  <dig> rust qtl were compared to the location of  <dig> qtl for stay-green reported in seven previous studies  <cit> , there was significant co-location across traits  with over half of the  <dig> rust qtl co-locating with stay-green qtl.
the highly significant qtl coincidence between rust and stay-green suggests many qtl impacting on both traits through a pleiotropic effect on carbon mobilisation.
rust qtl that are not associated with maturity and height are enriched for defence-related gene families
gene families associated with defence and disease resistance have previously been reported; the nbs-lrr gene family being the most prevalent and ancient and one of the largest gene families known in plants to be involved in the detection and response to diverse pathogens, including bacteria, viruses, fungi, nematodes, insects and oomycetes  <cit> .
the current study found that rust resistance qtl that were unique, and did not co-locate with either height, maturity or stay-green qtl , were significantly enriched for defence-related gene families , in contrast to the rust resistance qtl with potential pleiotropic effect that co-located with either height, maturity or stay-green qtl  .
some sorghum rust qtl fall within hotspots for multiple disease resistance
high correlations between resistances to rust and other biotic stresses have been reported previously in sorghum such as the positive correlation between resistance to rust, target leaf spot, zonate leaf spot and anthraconose observed by  <cit> .
additionally, hot-spots in the genome for multiple disease resistances are present in sorghum, particularly the short arm of sbi- <dig> and the long arm of sbi- <dig>  containing  <dig> qtl for  <dig> traits from  <dig> additional studies, as well as the current study  <cit> .
these hotspots could be the result of genes which provide resistance to multiple diseases, clusters of resistance genes or physiological pleiotrophy driven by regulation of carbon mobilisation.
of the  <dig> significant and suggestive rust qtl reported in the current study, over three quarters  co-located with  <dig> sorghum pest and disease resistance qtl from  <dig> studies previously projected onto the consensus map  <cit>  .
particular types of pest and disease resistance qtl were more likely to co-locate with the rust qtl associated with physiological pleiotrophic effects  while the reverse was true of fungal pathogens of the leaf  which predominantly co-locating with the classical rust resistance qtl, which did not co-locate with qtl associated with carbon stress.
the two stand-out hotspot regions identified for multiple disease resistances on sbi- <dig> and sbi- <dig> contained nbs-lrr gene clusters.
our result is consistent with three previous studies in sorghum that reported nbs-lrr genes conferring resistance to fungal pathogens; st-r gene cluster conferring resistance to setosphaeria turcica causing northern leaf blight in maize  <cit> , cs <dig> and cs <dig> genes conferring resistance to colletotrichum sublineolum causing anthracnose  <cit>  and rph1- <dig> conferring resistance to leaf rust  <cit> .
CONCLUSIONS
this study has dissected the genetic architecture of rust resistance in sorghum, through conventional qtl mapping and association mapping, with multiple rust resistance qtl of small effect identified.
using multiple lines of evidence, including colocation of qtl, direction of qtl effects and disease resistance gene family enrichment, we were able to classify  <dig> rust qtl identified in the current study as classical rust resistance qtl, and  <dig> rust qtl as multi-trait effect qtl where susceptibility is likely to have resulted from genes that influence changes in the plant carbon mobilisation during the reproductive phase.
the results of this study have significant implications for breeding programs attempting to increase disease resistance, particularly using molecular breeding approaches, because of the danger of inadvertently imposing selection pressure on associated traits, such as height and maturity, rather than disease resistance per se.
the genetic architecture of rust resistance in sorghum contrasts to rust resistance genes of large effect identified in other cereals, such as wheat, barley and oats, where rust epidemics frequently have more severe yield costs.
the depletion of plant carbon in the determinate annual winter cereal species, in contrast to the increased tendency towards perenniality in sorghum, could impose more severe carbon stress in wheat, barley and oats.
understanding of how senescence or other drivers of carbon remobilisation increase susceptibility to leaf diseases may provide a useful avenue for future work on developing novel approaches to controlling these diseases in other cereals.

 agriculture is facing enormous challenges to feed a growing population in the face of rapidly evolving pests and pathogens. a critical component for addressing these challenges is to breed for increased disease resistance in crop species to avoid the need for costly and potentially environmentally damaging pesticides. the major cereal crops feed over two thirds of the world’s population and yet the production of these crops continues to be challenged by pests and diseases with at least 30% of global food production lost to pathogens. sorghum moench) is a c cereal grain crop that provides staple food for over million people in the semi-arid tropics of africa and asia, in addition to being an important source of feed for livestock. amongst the cereals, sorghum is one of the best adapted to drought and high temperatures, and will play an increasingly important role in meeting the challenges of feeding the world’s growing population. in recent times, sorghum has become an attractive feedstock alternative for use in the production of bio-ethanol fuel. however, its productivity is often jeopardised by foliar fungal diseases. among the fungal diseases, leaf rust causes significant crop damage in sorghum, with rust diseases being among the most widespread and economically important diseases of cereals world-wide, e.g.. sorghum rust, caused by puccinia purpurea cooke, is widely distributed and occurs in almost all sorghum growing areas of the world. leaf rust frequently predisposes sorghum to other major diseases and agronomic problems, such as the fusarium stalk rots, charcoal rot, grain mould, anthracnose and lodging. in addition to indirect yield losses through predisposition to other diseases, sorghum leaf rust can cause direct yield losses of up to 50% depending on favourable environmental conditions for disease development and cultivar susceptibility. in australia, leaf rust has the most significant impact on grain yield of all fungal leaf diseases, causing up to 13% yield losses. the disease is seen every year on most sorghum crops, usually after flowering, when the leaves may be covered by the uredinia and telia of the fungus. due to the influence of environmental and climatic conditions on the development of the disease and the cost of fungicides, genetic resistance offers the best long-term solution for the management of leaf rust in sorghum. as such, better understanding of the genetic control of leaf rust resistance would provide sorghum breeders with critical knowledge to develop new resistant varieties. the genetic architecture of complex traits is frequently controlled by multiple genes or alleles that vary with genetic background. a single mapping population study can therefore explain only a small part of the global genetic architecture of a complex trait and limits the identification of potential genomic regions due to the absence or monomorphic presence of alleles contributing to the genetic control of a complex trait such as leaf rust resistance. initial studies into the inheritance of leaf rust resistance in sorghum identified a single dominant gene, pu, conferring rust resistance in sweet sorghum crosses. however, subsequent studies identified rust resistance to be polygenic in nature, with multiple genes and genomic regions identified. therefore, the comparison of qtl detected in multiple populations and multiple environments is particularly important for dissecting the genetic control of polygenic disease resistance and permits investigation of the degree to which the underlying genes contribute to variation in the phenotype under different genetic backgrounds and environmental conditions. furthermore, traits associated with the physiological development and age of the plant have been implicated in indirectly impacting disease response, with previous studies demonstrating that growth stage can impact on the degree of disease susceptibility in a range of crops e.g.. therefore, a multi-trait analysis approach enables the investigation of potentially pleiotropic disease response qtl. in this study, a combination of conventional qtl analysis and genome wide association analysis was used to determine the genetic architecture of leaf rust resistance in sorghum using three bi-parental mapping populations and a set of elite breeding lines phenotyped in hybrid combination with multiple testers. all populations were phenotyped not only for rust infection response, but also for height and maturity. further, the availability of the sorghum whole genome sequence and genetic linkage map-based resources, e.g., provided opportunities to compare qtl for rust resistance identified in the current study with previously reported qtl in both sorghum and maize, in addition to previously reported gene families associated with disease resistance.  phenotypic data variability the predicted means, ranges, and standard deviations for the traits measured for the progeny of the two ril populations are detailed in table  rust infection responses of the five parents in the three bi-parental populations are presented in additional file 1: figure s heritability for rust resistance was high in all three bi-parental populations. in the s population, the female parent had higher levels of rust resistance than the male parent; versus in the s population, the male parent exhibited partial resistance to rust, in contrast to the high level of susceptibility exhibited by the female parent. for s the rust resistant parent icsv was much later maturing and much taller than r between trait correlation was low. for s r931945-2- was slightly later maturing and shorter than is the rils showed transgressive segregation for all three traits. the predicted means, ranges, and standard deviations for all three traits scored in the s population are detailed in table  the s. bicolor subsp. verticilliflorum parent could not be grown in field trials because of its weedy nature. the predicted means, ranges, and standard deviations for the ayt association mapping set across the male testers and location combinations are detailed in additional file 2: table s heritability for rust resistance was found to be higher at the liverpool plains site, in comparison to the dalby site. the relative rust pressure was found to be reduced at the dalby site, in comparison to the other trials included in the study, through the comparison of rust response scores of check genotypes. the trials were not highly correlated; genotype × environment interaction was observed using a second order factor analytic site structure where only 68% of the variation was accounted for by the first factor, and additionally correlations across sites and within each male tester genotypes were higher than on a per site basis.table predicted mean values of rust infection score, height and maturity for the two ril populations and parental lines in the hrs trial, plus the standard deviation and range for each population population trait x¯parenta x¯parentb x¯progeny σ progeny min progeny max progeny 1 f 4 population across multiple trials, plus the standard deviation and range trial trait x¯progeny σ progeny min progeny max progeny bil2003irr bil2003nonirr bil2003overall bil2003irr bil2003nonirr bil2003overall bil2003irr bil2003nonirr bil2003overall qtl analysis the results of the qtl analysis for each trait in each population are shown in tables  and.table summary of rust resistance, maturity and height qtl identified in the s population, detailing the qtl position, 2-lod confidence interval, flanking markers, peak lod value, total trait variance explained, additive effect, and significance level qtl id lg peak cm a ci flanking markers lod r 2 b additive c sig d apeak position in cm based on the s genetic linkage map; bthe amount of total trait variance explained by a qtl at this locus, as %; cthe allelic effects are calculated as the effect of substitution of aa allele by bb allele; d*suggestive; **significant; ***highly significant. summary of rust resistance, maturity and height qtl identified in the s population, detailing the qtl position, 2-lod confidence interval, flanking markers, peak lod value, total trait variance explained, additive effect, and significance level qtl id lg peak a ci flanking markers lod r 2b additive c sig d apeak position in cm based on the s genetic linkage map; bthe amount of total trait variance explained by a qtl at this locus, as %; cthe allelic effects are calculated as the effect of substitution of aa allele by bb allele; d*suggestive; **significant; ***highly significant. summary of rust resistance qtl identified in the s population detailing the qtl location on consensus map, additive effect and significance level across sites allele effects b qtl id lg peak cm a bil03 irr bil03 nonirr bil03 overall apeak position with maximum –log10p; bthe allelic effects are calculated as the effect of substitution of aa allele by bb allele. ns: not significant; **significant; ***highly significant. rust resistance in population s cim identified two highly significant rust resistance qtl, one on sbi- and one on sbi-08-ii. in addition, three suggestive qtl were detected on sbi- sbi-06-i and sbi-08-i. individual qtl explained between to % of phenotypic variation in response to rust, with a cumulative total of %. the majority of qtl had positive allelic effects indicating that the icsv qtl alleles predominately contributed to an increase in rust resistance. in population s a total of qtl for rust resistance were identified located on chromosomes. one highly significant and three significant qtl were identified by cim analysis on sbi- sbi- sbi- and sbi-. five suggestive qtl were identified on sbi- sbi- sbi- and sbi- individual qtl explained between to % of phenotypic variation, with a cumulative total of %. six of the rust resistance qtl had negative effects, indicating that parent r931945-2- qtl alleles predominately contributed to an increase in rust resistance. in population s fifteen genomic regions were detected with significant marker trait associations on eight chromosomes. a further four genomic regions were detected with highly significant marker trait associations. the majority of the identified qtl had negative allele effects indicating that the s. bicolor subsp. verticilliflorum qtl alleles predominantly contributed to an increase in rust resistance. in the ayt association mapping set, genomic regions were identified with suggestive marker trait associations in at least one of the tester/location combinations, with over half identified as significant in two or more of the tester/location combinations. to combine the results of the association mapping analyses across the six male tester and location combinations, the number of tester/location combinations with a significant marker trait association was calculated for each marker and plotted against the sorghum consensus map based on a sliding window of 2 cm with a step size of  cm. of the qtl identified, were identified in a single tester/location combination only and hence can be considered as suggestive qtl regions. just over 10% of the qtl were influenced by the genetic background, being identified only with specific male testers across both locations. a further qtl were location-specific, being identified only in one location, however these included the suggestive qtl only identified in a single tester/location combination. three qtl were identified in all three male tester combinations at a single site only. a further three qtl were identified in both sites and across all male tester combinations. the total number of qtl identified with the male tester r986087-2-4- across both locations, was almost 50% higher than with either of the other two male testers, r and r995248; qtl vs qtl vs qtl respectively. the male line, r986087-2-4- was produced from a cross between r931945-2- and sc170-6-17; r931945-2- being a parental line of s and s populations. of the rust resistance qtl identified in both s and s were derived from r931945-2- the graphical genotype of r986087-2-4- based on previously generated gbs data indicated that r986087-2-4- was identical by descent to r931945-2- in of these qtl regions. in all of these regions, a qtl was also identified in the ayt populations with the r986087-2-4- tester.figure rust resistance qtl from the current study projected onto the sorghum consensus genetic linkage map in comparison with rust qtl identified in previous studies. the bars to the right of each chromosome indicate rust resistance qtl identified, colour-coded as follows: red: s; dark green: s; dark blue: s; green:; orange;; pink:. the locations of the qtl identified in the ayt association mapping set are represented by a separate heat map, for each lg, detailing qtl density. maturity in the s population, five suggestive qtl for maturity were identified located on four chromosomes, individually explaining between to % of the phenotypic variation, with a cumulative total of %. the maturity qtl on sbi- co-located with the highly significant rust resistance qtl in the same population, with the r alleles contributing towards lateness in maturity and rust resistance. in the s population, two highly significant qtl on sbi- and sbi- were identified, with a cumulative total of % of the phenotypic variation explained. three additional suggestive qtl were identified on sbi- sbi- and sbi- individually explaining between to % of the phenotypic variation. none of the maturity qtl co-located with rust qtl in the same population. both positive and negative additive effects were observed, indicating that the r931945-2- parent had alleles contributing to both an increase and a decrease in maturity. in s highly significant qtl were located on eight chromosomes. fourteen of the qtl had positive allele effects indicating that the s. bicolor subsp. verticilliflorum qtl alleles predominantly contributed to an increase in late maturity. a further two significant qtl regions were identified, both of which had positive allele effects. overall, approximately a quarter of the maturity qtl, four highly significant and one significant, in s co-located with rust qtl in the same population. the direction of allelic effects was consistent across co-locating qtl, with the rust resistance qtl alleles from s. bicolor subsp. verticilliflorum always occurring with late maturing alleles from s. bicolor subsp. verticilliflorum. in the ayt association mapping set, qtl for maturity were identified, eight of which co-located with rust qtl. height in the s population, one significant qtl on sbi- and three suggestive qtl on sbi- sbi- and sbi- were identified, with a cumulative total of % of phenotypic variation explained. three of the four qtl had negative allele effects, indicating that the qtl alleles from r predominantly contributed to a decrease in height. the height qtl on sbi- co-located with a suggestive rust qtl on sbi- in the same population, and the direction of qtl allele effects were consistent. in the s population, three highly significant qtl for height were identified, with a cumulative total of % of the phenotypic variation explained, on chromosomes sbi- sbi- and sbi-. three further suggestive qtl were identified on sbi- sbi- and sbi- the suggestive qtl for height on sbi- co-located with a rust qtl in the same population, while the highly significant height qtl on sbi- co-located with a maturity qtl. in both cases the direction of qtl allele effects was consistent with the observations from the s population. in s only one highly significant qtl for height was identified on sbi- while a further significant qtl were identified on sbi- sbi- sbi- and sbi-. five of the height qtl identified had positive allele effects indicating that the s. bicolor subsp. verticilliflorum qtl alleles predominantly contributed to an increase in height. the significant qtl for height on sbi- co-located with qtl for both rust and maturity, while the significant qtl for height on sbi- co-located within 10 cm a rust qtl. as previously, the direction of allele effects of the co-locating qtl was consistent with observations from the s and s populations. in the ayt association mapping set, qtl for height was identified on sbi- which co-located with rust and maturity qtl also identified in the ayt population. stay-green due to its association with carbon stress, the stay-green trait was also analysed in the ayt association mapping set. it was visually scored as a leaf senescence rating from 1–, across the same three male tester genotypes and two sites, with one trial site in common with the previous analyses, and analysed using the same methodology as described for the other traits, identifying nine significant qtl. qtl projection on consensus map all the qtl identified in each of the three bi-parental populations and the ayt association mapping set were projected on the consensus map for all three traits and compared to rust, maturity and height qtl identified previously. across the four populations in this study, significant or highly significant qtl and suggestive qtl for rust resistance were identified and projected onto the consensus map, in addition to the previously reported qtl for rust resistance, representing a total of unique regions. rust qtl were identified on all chromosomes, however the genome-wide distribution was uneven, with only unique qtl identified on sbi- in contrast to rust qtl located on sbi- the syntenic locations of rust qtl identified in maize were also projected onto the consensus map and compared to the locations of the rust qtl from sorghum. ten of the previously reported sorghum rust qtl co-located with rust qtl identified in the current study; one genomic region described by was found to be unique among the sorghum rust studies investigated, however this qtl co-located with maize rust qtl. in total, of the rust resistance qtl identified in maize, over 80% co-located with rust qtl identified in sorghum. the unique rust qtl regions identified were further categorised according to their co-location with height and maturity qtl, to distinguish between 1) qtl unique to rust within a population and 2) multi-trait effect qtl, with rust qtl co-locating with qtl for other traits within a population. of the unique genomic regions, were categorised as being unique to rust, whereas were not unique to rust, and co-located with either height, maturity or stay-green qtl identified in the same population. the correspondence of the two different types of rust resistance qtl locations and the nbs-lrr gene family members, previously reported to be associated with disease resistance, were investigated. the nbs-encoding genes were significantly enriched in the regions of the genome containing unique rust disease resistance qtl, in contrast to the rust qtl that co-located with other traits. in particular, the major nbs-encoding rust resistance gene rph1- homologous with the maize rp1-d gene, previously described in sorghum by and also believed to be the pu gene, co-located with qtl from the s population and the ayt association mapping set. additionally, the wheat rust resistance gene, lr previously described by, co-located with qtl from the s population and the ayt association mapping set, in addition to rust qtl identified in maize. both parental genotypes of the s and s populations were included in the recently described sorghum resequencing data set, enabling sequence analysis of underlying candidate genes. in silico analysis for missense mutations revealed a polymorphism leading to an amino-acid change that departed from the peptide arising from the predicted gene models in the candidate gene for lr which co-located with a rust resistance qtl in the s population. the thirty-eight qtl for maturity identified in s s s and the ayt association mapping set were projected onto the consensus map and compared to maturity qtl reported previously. of the maturity qtl identified in the current study, co-located with maturity qtl described previously. of the remaining qtl, co-located across the populations included in the current study, with only novel maturity qtl identified. two major-effect maturity loci previously projected onto the sorghum consensus map were identified in the current study; ma was identified in s, and ma was identified in populations s and s. similarly, the seventeen qtl for height identified in s s s and the ayt association mapping set were projected onto the consensus map and compared to height qtl reported previously. sixteen of the height qtl identified co-located with height qtl previously described, including the major effect height loci previously projected onto the consensus map; dw on sbi-, dw on sbi- and dw on sbi-. discussion the rusts are a major pathogen of cereal crops with the potential to cause large reductions in yield and have as a result been the focus of ongoing plant breeding effort particularly in the winter cereals. the challenge for breeders in these species has been to produce stable resistance due to the rapidly evolving nature of the pathogen. sorghum is a major summer cereal crop that is also a host for a rust pathogen puccinia purpurea. leaf rust occurs in almost all sorghum growing areas of the world and can cause significant yield losses in the crop. in australia, rust infection occurs in most sorghum crops with the severity of epidemics being greater in humid environments and in late-sown crops. despite the prevalence of the disease in sorghum, it is not subject to the large damaging rust epidemics which regularly occur in winter cereals in australia. understanding the genetic basis of rust in sorghum will be important for improving the crop itself as well as providing a comparison and potential insight into the architecture of the trait in other species. this study used natural field infection data from multiple populations grown in multiple environments using a combination of conventional linkage mapping and association mapping to investigate the genetic control of rust resistance and the potential for multi-trait effect qtl. rust resistance is a polygenic trait controlled by multiple qtl of small effect the complex, polygenic nature of this trait was confirmed with significant or highly significant qtl identified representing over unique genomic regions, in addition to suggestive qtl representing a further unique genomic regions. comparisons with previous rust resistance qtl studies revealed that of the previously reported rust resistance qtl in sorghum were also identified in the current study. the genetic architecture of the trait was largely found to involve multiple qtl of small effects, rather than major genes of large effect, with the largest allele effect reported in the s population, representing % of the total phenotypic variation, and with an average qtl allele effect across all qtl identified of just. this contrasts to the major effect leaf rust qtl commonly reported in other cereals, e.g. the major effect adult plant resistance gene rph in barley, where the additive allele effect sizes accounted for 64-85% of the phenotypic variation in adult plants across four field environments; the major effect, “slow-rusting” resistance gene lr conferring durable adult plant resistance in wheat, explaining up to 55% of the phenotypic variation. this difference in the genetic control of leaf rust resistance across cereals could be partly due to the fact that sorghum is primarily a perennial species in comparison to the strongly annual life-history of the other major cereals, where the need for more stable resistance due to increased plant longevity could favour selection for multiple resistance genes of small effect. this study encompassed populations with a degree of shared ancestry, which enabled the evaluation of resistance alleles that were identical by descent in different populations and experiments. the elite line r931945-2- was not only a common parent in the s and s populations but also a parent of the common male tester r986087-2-4- used to produce of the f hybrids used in the association mapping set. qtl alleles from r931945-2- contributed to increased rust resistance in just under half of the qtl identified from the two bi-parental populations; of these qtl, seven corresponded to genomic regions that were ibd between r986087-2-4- and r931945-2- in all of these regions, a qtl was identified in this location in the ayt association mapping set, in hybrids with r986087-2-4- as the common tester. this result suggests that the inheritance of these seven resistance qtl is not completely dominant, but instead either additive or recessive. this conclusion is also supported by the detection of the vast majority of the significant qtl in the ayt association mapping set identified across multiple male tester hybrid combinations. in commercial agricultural settings sorghum cultivars are almost exclusively f hybrids and as result knowledge of the degree of dominance in traits is of critical importance for their deployment as cultivars. the ibd analysis also enabled us to determine that two of the r931945-2- derived rust resistance qtl were identical to rust resistance qtl contributed by ql in the mapping population used by.the stability of these qtl suggests that there has been limited variation in the rust pathotypes over the last 15 years. in addition the fact that the majority of the rust qtl was detected at both ayt trial locations suggests that there is relatively low spatial and temporal variation in rust populations. rust resistance is strongly influenced by the physiological state of the plant in sorghum, rust infection generally develops later in the crop’s cycle as the plant begins filling grain. this is consistent with the observation of who demonstrated that the growth stage, or physiological age, of groundnut can impact on the degree of susceptibility to rust infection. typically plants in vegetative stages are less susceptible to many diseases than those infected at reproductive phases, when the plant is remobilizing carbohydrates during grain-filling. this phenomenon has important implications for assessing rust resistance in plants that vary in maturity or other characters such as height or stay-green which influence the developmental status of the plant and hence the nature and degree of carbohydrate remobilization. this is particularly important in field grown plants subject to natural infection. although the association between height and rust response could be related to a variation in microclimate, the association is consistent with the change in source/sink dynamics where earlier maturing plants become vulnerable to infection before later plants and taller plants have greater reserves for remobilization. carbon stress also plays an important role in drought responsiveness. plants with the stay-green characteristics have increased functional carbohydrate resources for remobilisation during grain filling. we observed low but consistent, negative phenotypic correlations between the severity of rust infection and both maturity and height. in all populations and trials early maturity and short stature was associated with rust susceptibility. this result is consistent with the hypothesis that variation in plant carbohydrate status influences susceptibility to rust. at the qtl level we found a highly significant coincidence of rust qtl with qtl for height and maturity in the four populations used. a statistically significant proportion of the rust qtl co-located with one of these traits in the same population. a recent synthesis of disease resistance qtl studies in maize found comparable significant associations between disease resistance qtl and maturity qtl in the maize genome. co-locating qtl regions could contain multiple tightly linked genes for resistance and other traits or single genes with a pleiotropic effect across multiple traits. however the evidence for pleiotropic qtl action in the current study is very strong given the consistency in the direction of qtl allele effects, i.e. taller and later maturing plants carried the rust resistant qtl allele in the majority of cases. this is consistent with a recent study analysing the genetic control of rust in sorghum who found rust severity in field grown sorghum was negatively correlated with plant height and maturity. interestingly, when the same genotypes were artificially infected at a single time point in the glasshouse these correlations were not apparent. this provides further evidence that the physiological state influences either the timing of infection or the rate at which infection develops. the co-location of 100% of the leaf senescence, stay-green characteristic, qtl identified in the ayt association mapping set with rust qtl, in addition to the consistency of the direct of the effects, provides further evidence to support the role of carbon stress in disease resistance. additionally, when all rust qtl were compared to the location of qtl for stay-green reported in seven previous studies, there was significant co-location across traits with over half of the rust qtl co-locating with stay-green qtl. the highly significant qtl coincidence between rust and stay-green suggests many qtl impacting on both traits through a pleiotropic effect on carbon mobilisation. rust qtl that are not associated with maturity and height are enriched for defence-related gene families gene families associated with defence and disease resistance have previously been reported; the nbs-lrr gene family being the most prevalent and ancient and one of the largest gene families known in plants to be involved in the detection and response to diverse pathogens, including bacteria, viruses, fungi, nematodes, insects and oomycetes. the current study found that rust resistance qtl that were unique, and did not co-locate with either height, maturity or stay-green qtl, were significantly enriched for defence-related gene families, in contrast to the rust resistance qtl with potential pleiotropic effect that co-located with either height, maturity or stay-green qtl. some sorghum rust qtl fall within hotspots for multiple disease resistance high correlations between resistances to rust and other biotic stresses have been reported previously in sorghum such as the positive correlation between resistance to rust, target leaf spot, zonate leaf spot and anthraconose observed by. additionally, hot-spots in the genome for multiple disease resistances are present in sorghum, particularly the short arm of sbi- and the long arm of sbi- containing qtl for traits from additional studies, as well as the current study. these hotspots could be the result of genes which provide resistance to multiple diseases, clusters of resistance genes or physiological pleiotrophy driven by regulation of carbon mobilisation. of the significant and suggestive rust qtl reported in the current study, over three quarters co-located with sorghum pest and disease resistance qtl from studies previously projected onto the consensus map. particular types of pest and disease resistance qtl were more likely to co-locate with the rust qtl associated with physiological pleiotrophic effects while the reverse was true of fungal pathogens of the leaf which predominantly co-locating with the classical rust resistance qtl, which did not co-locate with qtl associated with carbon stress. the two stand-out hotspot regions identified for multiple disease resistances on sbi- and sbi- contained nbs-lrr gene clusters. our result is consistent with three previous studies in sorghum that reported nbs-lrr genes conferring resistance to fungal pathogens; st-r gene cluster conferring resistance to setosphaeria turcica causing northern leaf blight in maize, cs and cs genes conferring resistance to colletotrichum sublineolum causing anthracnose and rph1- conferring resistance to leaf rust.  this study has dissected the genetic architecture of rust resistance in sorghum, through conventional qtl mapping and association mapping, with multiple rust resistance qtl of small effect identified. using multiple lines of evidence, including colocation of qtl, direction of qtl effects and disease resistance gene family enrichment, we were able to classify rust qtl identified in the current study as classical rust resistance qtl, and rust qtl as multi-trait effect qtl where susceptibility is likely to have resulted from genes that influence changes in the plant carbon mobilisation during the reproductive phase. the results of this study have significant implications for breeding programs attempting to increase disease resistance, particularly using molecular breeding approaches, because of the danger of inadvertently imposing selection pressure on associated traits, such as height and maturity, rather than disease resistance per se. the genetic architecture of rust resistance in sorghum contrasts to rust resistance genes of large effect identified in other cereals, such as wheat, barley and oats, where rust epidemics frequently have more severe yield costs. the depletion of plant carbon in the determinate annual winter cereal species, in contrast to the increased tendency towards perenniality in sorghum, could impose more severe carbon stress in wheat, barley and oats. understanding of how senescence or other drivers of carbon remobilisation increase susceptibility to leaf diseases may provide a useful avenue for future work on developing novel approaches to controlling these diseases in other cereals. 
the distinction of disease resistance qtl hot-spots, enriched with defence-related gene families from qtl which impact on development and partitioning, provides plant breeders with knowledge which will allow for fast-tracking varieties with both durable pathogen resistance and appropriate adaptive traits. comparisons across populations within the current study and with rust qtl identified previously in both sorghum and maize revealed a high degree of correspondence in qtl location. sorghum is a major summer cereal crop that is also a host for a rust pathogen puccinia purpurea, which occurs in almost all sorghum growing areas of the world, causing direct and indirect yield losses in sorghum worldwide, however knowledge about its genetic control is still limited. in order to further investigate this issue, qtl and association mapping methods were implemented to study rust resistance in three bi-parental populations and an association mapping set of elite breeding lines in different environments. in total, significant or highly significant qtl and suggestive rust resistance qtl were identified representing unique genomic regions. classical rust resistance qtl regions that did not co-locate with height, maturity or stay-green qtl were found to be significantly enriched for the defence-related nbs-encoding gene family, in contrast to the lack of defence-related gene enrichment in multi-trait effect rust resistance qtl. rust resistancesorghumpleiotropyheightmaturitystay-greenqtl mappingassociation mappingissue-copyright-statement© the author 2014 negative phenotypic correlations were observed between rust, maturity and height, indicating a trend for both early maturing and shorter genotypes to be more susceptible to rust. the significant amount of qtl co-location across traits, in addition to the consistency in the direction of qtl allele effects, has provided evidence to support pleiotropic qtl action across rust, height, maturity and stay-green, supporting the role of carbon stress in susceptibility to rust. the rusts, in particular, are a major pathogen of cereal crops with the potential to cause large reductions in yield.
 agriculture is facing enormous challenges to feed a growing population in the face of rapidly evolving pests and pathogens. the rusts, in particular, are a major pathogen of cereal crops with the potential to cause large reductions in yield. improving stable disease resistance is an on-going major and challenging focus for many plant breeding programs, due to the rapidly evolving nature of the pathogen. sorghum is a major summer cereal crop that is also a host for a rust pathogen puccinia purpurea, which occurs in almost all sorghum growing areas of the world, causing direct and indirect yield losses in sorghum worldwide, however knowledge about its genetic control is still limited. in order to further investigate this issue, qtl and association mapping methods were implemented to study rust resistance in three bi-parental populations and an association mapping set of elite breeding lines in different environments.  in total, significant or highly significant qtl and suggestive rust resistance qtl were identified representing unique genomic regions. comparisons across populations within the current study and with rust qtl identified previously in both sorghum and maize revealed a high degree of correspondence in qtl location. negative phenotypic correlations were observed between rust, maturity and height, indicating a trend for both early maturing and shorter genotypes to be more susceptible to rust.  the significant amount of qtl co-location across traits, in addition to the consistency in the direction of qtl allele effects, has provided evidence to support pleiotropic qtl action across rust, height, maturity and stay-green, supporting the role of carbon stress in susceptibility to rust. classical rust resistance qtl regions that did not co-locate with height, maturity or stay-green qtl were found to be significantly enriched for the defence-related nbs-encoding gene family, in contrast to the lack of defence-related gene enrichment in multi-trait effect rust resistance qtl. the distinction of disease resistance qtl hot-spots, enriched with defence-related gene families from qtl which impact on development and partitioning, provides plant breeders with knowledge which will allow for fast-tracking varieties with both durable pathogen resistance and appropriate adaptive traits. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rust resistancesorghumpleiotropyheightmaturitystay-greenqtl mappingassociation mappingissue-copyright-statement© the author 2014 