BACKGROUND
chickpea  or garbanzo beans  is the second largest cultivated legume crop after dry beans globally  <cit> .
it is grown in  <dig> countries as a rainfed, post-rainy season and winter crop in subtropical south asia, parts of africa and australia and as a spring season crop in the temperate and mediterranean regions  <cit> .
during  <dig>  chickpea covered a total of  <dig>  million ha  area worldwide with a global production of  <dig>  million tons  and average productivity of  <dig>  kg ha− <dig>  where as in india it covered  <dig>  m ha with the production of  <dig>  m t and average yield of 912 kg ha− <dig> <cit> .
india is the largest producer of chickpea and accounts for  <dig> % of the total area and  <dig> % of total production globally.
chickpea represents  <dig> % of total pulse area and  <dig> % of total pulse production in india  <cit> .
various biotic and abiotic stresses affect stable and high yields of chickpea crop worldwide.
among the biotic stresses, fusarium wilt , caused by the asexual, soil borne and seed borne fungus fusarium oxysporum schlecht and emnd snyd.
& hans.
f. sp.
ciceris  snyd.
and hans.
, results in major economic losses ranging from 10-40% worldwide.
it is estimated to cause 10-15% yield loss annually in india  <cit> , but can result in 100% losses under favourable conditions.
the pathogen enters host through roots and causes systemic infection by progressive vascular damage, wilting and finally death of the plant.
the cultivation of resistant varieties is one of the most durable and economical practice for the management of fw.
however, performance of varieties differs from place to place owing to existence of physiological races among the foc isolates.
eight races of foc  have been reported worldwide .
races 1a ,  <dig>   <dig> and  <dig> have been reported from india, whereas races  <dig>  1b/c,  <dig> and  <dig> were found mainly in the mediterranean region and in the united states .
among all the races, race  <dig> is more widespread and has been reported in india, california and the mediterranean region.
pathogen also exhibit race specific differences in disease symptomatology; the yellowing and wilting  <cit> , the first produces progressive yellowing of leaves and vascular discoloration and plant dies 40 days after inoculation of the pathogen, while later induces severe chlorosis, flaccidity and vascular discoloration and the plant dies 20 days after inoculation  <cit> .
races  <dig> and 1b/c induce yellowing symptoms, whereas the remaining races  induce wilting.
recently, change in the race scenario of foc has been reported by dubey et al.
<cit>  and sharma et al.
<cit>  in india.
therefore, the identification of pathogenic races of foc is important for disease resistance breeding and for the efficient use of available fw resistant cultivars in chickpea.
monitoring pathogenic variability of fungus based on dna markers will greatly help in understanding pathogen diversity and their pathogenicity.
in past, different kinds of molecular markers were employed to characterize foc isolates, for instance, random amplified polymorphic dna   <cit> , restriction fragment length polymorphism   <cit> , amplified fragment length polymorphism   <cit> , inter simple sequence repeats   <cit>  and simple sequence repeats   <cit> .
however, the marker systems are put to limited use because of lack of reliability and resolution  or labour intensive and not amenable for throughput genotyping  or initial development costs are very high or require sequence information .
in this context, diversity arrays technology  developed by jaccoud et al.
<cit>  is the best platform to generate thousands of markers in species like foc with meagre genomic resources.
this technology has been successfully employed in several crop plants for characterization of germplasm, trait mapping, and establishing marker trait associations .
the present study reports on the development of dart markers and characterization of foc isolates.
the specific objectives of this study were i) to develop high density foc dart arrays ii) to assess effectiveness of dart markers in diversity study on a set of  <dig> foc isolates representing diverse agro-ecological zones of india and iii) comparative assessment of genotypic and phenotypic data based on standard set of differential cultivars of chickpea.
this new molecular marker system for foc will serve as an additional resource to augment the existing systems to assist crop improvement efforts, disease management practices and genetic studies in chickpea.
RESULTS
virulence diversity
based on disease reaction of  <dig> isolates collected from five diverse chickpea growing agro-ecological zones like central zone , north east plain zone , north hill zone , north west plain zone  and south zone  representing  <dig> states and  <dig> locations in india  on a standard set of ten differentials, all the isolates showed a large variation in virulence .
the dendrogram based on virulence data on differentials grouped the isolates into three major clusters .
grouping of the isolates clearly indicated the existence of more than one race in a region.
presence of race  <dig> was observed irrespective of zones in all the clusters.
however, most of the isolates of race  <dig> reaction were grouped in cluster i. cluster ii represented the isolates with least virulent reaction irrespective of location and cluster iii was dominated by race  <dig>  the race reaction indicated the widespread occurrence of race  <dig>  followed by new race  <dig>  in india.
race  <dig> reactions were found only in  <dig> isolates  from uttar pradesh , maharashtra  and gujarat  and race  <dig> reactions in only one isolate from bihar  .
none of the isolates used in the present study had a reaction similar to race  <dig>  sixteen isolates were found with different disease reaction which did not match to any race reactions.
however, these isolates grouped together and their reaction was either susceptible or moderately resistant on jg  <dig>  and resistant to other differential cultivars.
disease reactions of standard chickpea differential cultivars against different isolates of foc are provided in additional file  <dig> table  <dig>
passport information of isolates used in
fusarium oxysporum
f. sp
.
ciceris
dart array development, phenotyping and genotyping
*isolates used for construction of dart array.
**ap - andhra pradesh, br - bihar, cg - chhattisgarh, dl - delhi, gj - gujarat, hr - haryana, hp - himachal pradesh, ka - karnataka, mp - madhya pradesh, mh - maharashtra, pb - punjab, up - uttar pradesh, uk - uttarakhand,
***cz - central zone, nepz - north east plain zone, nhz - north hill zone, nwpz - north west plain zone and sz - south zone.
dendrogram derived from virulence analysis of  <dig> isolates of
f. oxysporum
f. sp.
ciceris
by upgma with  <dig> chickpea differential cultivars.
dart array development and evaluation
a total of  <dig> foc isolates that represent whole genome diversity of foc were used for developing the diversity arrays using psti/hpaii restriction endonucleases.
a total of  <dig>  dart markers were developed from psti/hpaii restriction endonucleases combinations generated from a mixture of dna of  <dig> foc isolates.
of  <dig>  markers,  <dig>  markers were found polymorphic using dartsoft  <dig> .
the overall marker quality was high, with approximately > 90% call rate .
the call rate of the polymorphic markers ranged from 75-100% with an average of  <dig> % and had scoring reproducibility 100%.
the allele frequencies of polymorphic dart markers ranged from  <dig> - <dig>  .figure  <dig>
distribution of call rate among the  <dig>  dart markers.
average polymorphism information content  value for dart markers was  <dig> .
about  <dig> % of markers had pic value in the range of  <dig> - <dig> , while  <dig> % and  <dig> % markers were in the range of  <dig> - <dig>  and  <dig> - <dig> , respectively .
further,  <dig> % and  <dig> % of markers had the pic values in a range of  <dig> - <dig>  and  <dig> - <dig>  respectively.
overall the distribution of pic values was asymmetrical and skewed towards the higher value .
further, when the quality of the dart markers was analyzed against their performance, determined by the call rate and pic values,  <dig> % of the polymorphic dart markers  were in the 80-100% quality category with an average pic value of  <dig>  and a call rate of  <dig> % .
the average pic value increased with the average quality value.
the pic values for  <dig> markers possessing marker quality of <50% ranged from  <dig> - <dig> .
of  <dig> markers with a quality of more than 80%, the  <dig> markers had a pic value of > <dig> .
out of  <dig>  polymorphic dart markers in foc analyzed, there are  <dig> and  <dig> markers in the pic value range of  <dig> - <dig>  and  <dig> - <dig> , respectively .figure  <dig>
number of dart markers in different pic value classes.
genetic relationship among foc isolates
a total of  <dig> isolates of foc  were employed for gaining insights into the genetic diversity patterns.
using the  <dig>  polymorphic markers, upgma dendrogram was generated with the  <dig> isolates .
the estimated reliability of the branches of the dendrogram was also supported by the bootstrap values as shown in figure  <dig>  all the isolates tested could be divided into four major clusters with  <dig>   <dig>   <dig>   <dig> isolates clustered in cluster i, cluster ii, cluster iv and cluster iii respectively.
however, a total of  <dig> isolates were grouped separately from the above mentioned major clusters.
in each cluster, isolates from different agro-ecological zones was distributed.
however, certain region specific sub-grouping within each cluster was found.figure  <dig>
cluster analysis of
f. oxysporum
f. sp.
ciceris
isolates based on  <dig>  polymorphic dart markers.
cluster i represented 49% isolates from sz  and 34% from nwpz and nepz .
in the second cluster, 58% isolates were from nwpz and nhz  and 34% from cz .
the third cluster was smallest with the isolates mostly from nwpz and nepz  and 38% from cz .
fourth cluster represented 67% isolates from sz  and remaining from cz and nwpz .
few isolates  from ap grouped separately and clustered at the top of the dendrogram.
also few isolates were found to be isolated from major groups for instance isolates from br , hp , gj  and mp .the pcoa based on polymorphic dart marker data derived from  <dig> isolates, confirmed the four different groups found by upgma dendrogram .
the two dimensions of pcoa explained  <dig> % and  <dig> % of the variation present in the genetic distance calculated between isolates.
these two components were responsible for a total  <dig> % of the variation observed among the isolates.
the right-hand groups  proved to be clustered around the pcoa x- axis with isolates originating them different agro-ecological zone.
this group corresponded to the isolates from cluster iii of the upgma dendrogram.
the left hand groups were spread widely along both the pcoa x- axis and pcoa y- axis.
these groups represented the isolates from cluster-i, ii and iv of the upgma dendrogram.figure  <dig>
distribution of 110
f. oxysporum
f. sp.
ciceris
isolates in different quadrants based principal coordinate analysis using  <dig>  dart markers.
the principal coordinate i explain  <dig> % and principal coordinate ii explain  <dig> % variation.
population structure of foc
in order to assess the population structure and number of sub-populations,  <dig>  polymorphic dart markers were used on  <dig> foc isolates employing structure  <dig> .
<dig>  based on the maximum likelihood and delta k  values, four sub-populations were determined in  <dig> foc isolates .
each vertical bar in the figure represents a single foc isolate and its inferred proportion of genetic admixture.
the parts with different colours represent four different sub-populations.
using a membership probability threshold of  <dig> ,  <dig> isolates were assigned to sub-population  <dig>   <dig> isolate to sub-population  <dig>   <dig> isolates to sub-population  <dig> and  <dig> isolates to sub-population  <dig>  detailed information of sub-populations and respective isolate within each sub-population is provided in additional file  <dig>  sub-population  <dig>   <dig> and  <dig> have genetic admixture, however sub-population  <dig> have homogeneous genetic background.figure  <dig>
population structure among 110
f. oxysporum
f. sp.
ciceris
isolates  the highest delta k value is observed for k value  <dig>  indicating  <dig> sub-populations among the foc isolates used in the study  genetic composition of different sub-populations.
allele frequency
the allelic composition revealed the predominance of common  and most frequent alleles  in the total  <dig>  polymorphic alleles .
foc population contained a lower proportion of rare alleles.
among  <dig>  polymorphic markers, most frequent alleles were represented by  <dig> % and common alleles contributed to  <dig> %.
however,  <dig> unique alleles were detected in the foc isolates whose reaction did not match with any specific races.
further, race specific unique alleles were also identified for different race specific isolates for instance  <dig> ,  <dig>  and  <dig> .
discussion
the importance of understanding genetic diversity in any fungal species collection is critical for their effective management.
although tremendous progress has been made in terms of availability of molecular markers to study the genetic diversity in foc  <cit> ; availability of sound, reliable and cost-effective marker platform is still lacking.
in the present study, we present the first complete report on the development of dart marker platform for any fungal pathogen.
to the best of our knowledge, this is the first attempt to develop dart markers for foc and investigation on the utility of these markers in genetic diversity of large number of foc isolates collected from various regions in india.
we further demonstrated the pathogenic races, as determined by the disease reaction on differential cultivars-correlated to inferred dart genotyping.
the dart technology combines a reduction of genome complexity with high throughput and cost-effective hybridization based polymorphism detection.
the advantage of using these dart markers is that they can simultaneously type several thousand loci in a single array and provide a cost-effective and sequence independent tool for whole genome fingerprinting  <cit> .
the dart technology has been applied to a number of plants and animals .
however, no information is available on any plant pathogen till date.
in this study, the new dart platform for foc was found acceptable and provided robust information about the genetic diversity in this collection.
out of  <dig>  dart markers,  <dig>  markers were found to be useful in providing a complete picture of genetic diversity in the foc collection of  <dig> isolates.
overall, the average pic value for dart marker was found to be lower than that observed in other plant species , where similar markers were developed  <cit> , but comparable to that observed in pigeonpea  <cit>  and sugar beet  <cit> .
nearly nine per cent of dart markers have a pic value in the range of  <dig> - <dig>  and these markers therefore may be considered informative.
dart markers developed in this study effectively detected diversity among foc isolates.
all the three types of dart diversity analysis  indicated that the different isolates were successfully classified by the marker system and reflected high genetic diversity.
the phylogeny of the foc isolates inferred from dart markers by cluster analysis indicated  <dig> major clusters among them.
the subgroup clustering pattern with in each major cluster was consistent, indicating that the dart markers faithfully detected small differences among isolates.
a similar finding was reported earlier from different markers systems like rapd  <cit> , aflp  <cit>  and ssr  <cit>  with considerably less resolution and less transferability.
virulence analysis on a set of differential cultivars is required to authenticate the groups generated by the molecular markers.
in this study, virulence analysis based on standard set of differentials showed the existence of highly variable populations of foc in different agro-climatic zones in india.
the reactions on differential cultivars of chickpea indicated the existence of races  <dig>   <dig>   <dig> and  <dig> in india.
in addition few isolates  did not show match with any of the race reaction and was grouped separately in both the dendrograms.
previous studies based on differentials reported the presence of eight pathogenic races  in foc based on disease reactions on a set of differential chickpea cultivars  <cit> .
race  <dig> represented 47% of total foc isolates, of which most of the isolates grouped in cluster i. previous studies reported race  <dig> only from ap and ka states of india  <cit> , however present diversity based on dart indicated its presence in all the agro-ecological zones of india, with predominance in sz.
the high genetic diversity among the race  <dig> isolates of foc revealed its diverse geographical inhabitation under the present scenario.
this widespread distribution of race  <dig> can be attributed to the seed transmission through large exchange of germplasm that took place more in last one decade.
among all the races, race  <dig> is more widespread and has been reported in india, california, and the mediterranean region  <cit> .
race  <dig> represented 29% of total foc isolates, of which most of the isolates were grouped into cluster ii.
the race  <dig> was previously confined to mediterranean basin.
it was first reported from california  <cit>  later reported from spain  <cit> , israel and morocco  <cit> .
occurrence of this new race of foc in india mainly appeared to be a recent introduction through seed transmission and pathogen genetic variability from accumulation of mutations over the time.
according to jimenez et al.
, there may have little or no selection for resistance –breaking races of foc, minimizing the number of parallel events in acquisition of virulence, which are frequently observed in other system.
thus, unlike other pathosystems  there may have been little or no selection for resistance breaking races of foc, which minimizes the probabilities of obtaining parallel changes in virulence.
they further stated that it is not probable that the stepwise evolution of races in foc resulted from the selection by specific resistance in chickpea populations, as widespread use of race  <dig> a-resistant cultivars in india has not yet led to reports on development of race  <dig>  on the contrary, we speculate that stepwise evolution of races in foc resulted from selection by specific resistance in chickpea populations or repeated migration has occurred rather than the independent evolution of races in different regions or as expected from an ancient lineage race  <dig> could be the common ancestor of all races.
presence of race  <dig> in india is also indicated by dubey et al.
<cit> .
further, there are reports that races  <dig> and  <dig> have similar virulence pattern on chickpea differentials  <cit>  and high genetic similarities  <cit> .
earlier researchers indicated that it is possible that misclassification of these isolates occurred during pathogenicity test.
it is also possible that these two pathogenic races were not yet evolutionarily separated from each other to have developed diagnostic and separate pathogenicity test.
the isolates grouped in cluster iii represent those isolates which had susceptible reaction with less virulence on jg  <dig> cultivar and resistance reaction on all the other differential cultivars.
this group corresponded to 19% of total foc isolates representing nz.
difference in virulence among foc isolates originating from southern and northern partes of india has also been reported by dubey et al.
<cit> .
the distribution of virulence within and among populations of foc is probably the result of different selection pressure exerted by a specific resistant gene in the chickpea varieties cultivated in the area.
this is in agreement with the observations by lebeda and petrzelova  <cit>  in the case of bremia lactucae.
also, this could be the result of shift in the cultivation of chickpea from northern india to southern india  <cit> , which further supported the low frequency of race  <dig>  originally reported from kanpur, north india.
race  <dig> disease reaction was not found in any of the isolates.
the absence of race  <dig> in the present collection is in agreement with gurjar et al.
<cit> .
they found completely different reaction of race  <dig> and reported its resemblance to fusarium proliferatum based on phylogenetic analysis.
the isolates in cluster iv mainly represent multiple race reaction race  <dig>  race  <dig>  race  <dig> and race  <dig>  regardless of the origin, the similarities of race composition between the different zones of india indicated that repeated migration of infected seeds.
the unique, common and most frequent alleles among the total  <dig>  polymorphic markers reported in the present study are novel features.
such features, to the best of our knowledge, have not been reported in other diversity study.
race specific alleles detected in the present study will be very useful for molecular profiling of a particular race.
further, occurrence of common alleles will be useful for understanding the similarity and also molecular profiling of foc isolates from diverse geographical locations.
two rare alleles observed specific to up location could be useful in the detection of location specific foc isolates.
CONCLUSIONS
the present study generated significant information in terms of pathogenic and genetic diversity of foc which could be used further for deployment of region-specific resistant varieties of chickpea.
the study clearly highlighted the occurrence of multiple/new races of foc in india and suggests the continuous monitoring of changes in foc population to check the susceptibility of widely grown resistant cultivars against these races.
the dart markers developed in the present study are powerful diagnostic tools for understanding the diversity in foc population in chickpea.
in this study, we reported the successful development of dart marker platform for foc and their utility in genotyping of foc collections.
this dart marker system complemented the microsatellite markers developed previously for foc.
the high number of dart markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of variability in the foc population present in india, as well as provided support to know the changing race scenario in foc population.
further, the dart markers identified in this study could be used to study the distribution of these foc races, and to facilitate the efficient deployment of available host resistance.
these markers should assist in the early detection of introduced race, as well as of changes in the relative frequencies of different races that might occur in response to the use of resistant chickpea cultivars.
the dart arrays developed for foc in this study are available with the center of excellence in genomics  at icrisat.
also the foc isolates used in the present study are available with legumes pathology unit at icrisat for further use by any scientific community.

 chickpea or garbanzo beans is the second largest cultivated legume crop after dry beans globally. it is grown in countries as a rainfed, post-rainy season and winter crop in subtropical south asia, parts of africa and australia and as a spring season crop in the temperate and mediterranean regions. during chickpea covered a total of million ha area worldwide with a global production of million tons and average productivity of  kg ha− where as in india it covered  m ha with the production of  m t and average yield of 912 kg ha−. india is the largest producer of chickpea and accounts for % of the total area and % of total production globally. chickpea represents % of total pulse area and % of total pulse production in india. various biotic and abiotic stresses affect stable and high yields of chickpea crop worldwide. among the biotic stresses, fusarium wilt, caused by the asexual, soil borne and seed borne fungus fusarium oxysporum schlecht and emnd snyd. & hans. f. sp. ciceris snyd. and hans., results in major economic losses ranging from 10-40% worldwide. it is estimated to cause 10-15% yield loss annually in india, but can result in 100% losses under favourable conditions. the pathogen enters host through roots and causes systemic infection by progressive vascular damage, wilting and finally death of the plant. the cultivation of resistant varieties is one of the most durable and economical practice for the management of fw. however, performance of varieties differs from place to place owing to existence of physiological races among the foc isolates. eight races of foc have been reported worldwide. races 1a, and have been reported from india, whereas races 1b/c, and were found mainly in the mediterranean region and in the united states. among all the races, race is more widespread and has been reported in india, california and the mediterranean region. pathogen also exhibit race specific differences in disease symptomatology; the yellowing and wilting, the first produces progressive yellowing of leaves and vascular discoloration and plant dies 40 days after inoculation of the pathogen, while later induces severe chlorosis, flaccidity and vascular discoloration and the plant dies 20 days after inoculation. races and 1b/c induce yellowing symptoms, whereas the remaining races induce wilting. recently, change in the race scenario of foc has been reported by dubey et al. and sharma et al. in india. therefore, the identification of pathogenic races of foc is important for disease resistance breeding and for the efficient use of available fw resistant cultivars in chickpea. monitoring pathogenic variability of fungus based on dna markers will greatly help in understanding pathogen diversity and their pathogenicity. in past, different kinds of molecular markers were employed to characterize foc isolates, for instance, random amplified polymorphic dna, restriction fragment length polymorphism, amplified fragment length polymorphism, inter simple sequence repeats and simple sequence repeats. however, the marker systems are put to limited use because of lack of reliability and resolution or labour intensive and not amenable for throughput genotyping or initial development costs are very high or require sequence information. in this context, diversity arrays technology developed by jaccoud et al. is the best platform to generate thousands of markers in species like foc with meagre genomic resources. this technology has been successfully employed in several crop plants for characterization of germplasm, trait mapping, and establishing marker trait associations. the present study reports on the development of dart markers and characterization of foc isolates. the specific objectives of this study were i) to develop high density foc dart arrays ii) to assess effectiveness of dart markers in diversity study on a set of foc isolates representing diverse agro-ecological zones of india and iii) comparative assessment of genotypic and phenotypic data based on standard set of differential cultivars of chickpea. this new molecular marker system for foc will serve as an additional resource to augment the existing systems to assist crop improvement efforts, disease management practices and genetic studies in chickpea.  virulence diversity based on disease reaction of isolates collected from five diverse chickpea growing agro-ecological zones like central zone, north east plain zone, north hill zone, north west plain zone and south zone representing states and locations in india on a standard set of ten differentials, all the isolates showed a large variation in virulence. the dendrogram based on virulence data on differentials grouped the isolates into three major clusters. grouping of the isolates clearly indicated the existence of more than one race in a region. presence of race was observed irrespective of zones in all the clusters. however, most of the isolates of race reaction were grouped in cluster i. cluster ii represented the isolates with least virulent reaction irrespective of location and cluster iii was dominated by race the race reaction indicated the widespread occurrence of race followed by new race in india. race reactions were found only in isolates from uttar pradesh, maharashtra and gujarat and race reactions in only one isolate from bihar. none of the isolates used in the present study had a reaction similar to race sixteen isolates were found with different disease reaction which did not match to any race reactions. however, these isolates grouped together and their reaction was either susceptible or moderately resistant on jg and resistant to other differential cultivars. disease reactions of standard chickpea differential cultivars against different isolates of foc are provided in additional file table passport information of isolates used in fusarium oxysporum f. sp. ciceris dart array development, phenotyping and genotyping *isolates used for construction of dart array. **ap - andhra pradesh, br - bihar, cg - chhattisgarh, dl - delhi, gj - gujarat, hr - haryana, hp - himachal pradesh, ka - karnataka, mp - madhya pradesh, mh - maharashtra, pb - punjab, up - uttar pradesh, uk - uttarakhand, ***cz - central zone, nepz - north east plain zone, nhz - north hill zone, nwpz - north west plain zone and sz - south zone. dendrogram derived from virulence analysis of isolates of f. oxysporum f. sp. ciceris by upgma with chickpea differential cultivars. dart array development and evaluation a total of foc isolates that represent whole genome diversity of foc were used for developing the diversity arrays using psti/hpaii restriction endonucleases. a total of dart markers were developed from psti/hpaii restriction endonucleases combinations generated from a mixture of dna of foc isolates. of markers, markers were found polymorphic using dartsoft. the overall marker quality was high, with approximately > 90% call rate. the call rate of the polymorphic markers ranged from 75-100% with an average of % and had scoring reproducibility 100%. the allele frequencies of polymorphic dart markers ranged from -.figure distribution of call rate among the dart markers. average polymorphism information content value for dart markers was. about % of markers had pic value in the range of -, while % and % markers were in the range of - and -, respectively. further, % and % of markers had the pic values in a range of - and - respectively. overall the distribution of pic values was asymmetrical and skewed towards the higher value. further, when the quality of the dart markers was analyzed against their performance, determined by the call rate and pic values, % of the polymorphic dart markers were in the 80-100% quality category with an average pic value of and a call rate of %. the average pic value increased with the average quality value. the pic values for markers possessing marker quality of <50% ranged from -. of markers with a quality of more than 80%, the markers had a pic value of >. out of polymorphic dart markers in foc analyzed, there are and markers in the pic value range of - and -, respectively.figure number of dart markers in different pic value classes. genetic relationship among foc isolates a total of isolates of foc were employed for gaining insights into the genetic diversity patterns. using the polymorphic markers, upgma dendrogram was generated with the isolates. the estimated reliability of the branches of the dendrogram was also supported by the bootstrap values as shown in figure  all the isolates tested could be divided into four major clusters with isolates clustered in cluster i, cluster ii, cluster iv and cluster iii respectively. however, a total of isolates were grouped separately from the above mentioned major clusters. in each cluster, isolates from different agro-ecological zones was distributed. however, certain region specific sub-grouping within each cluster was found.figure cluster analysis of f. oxysporum f. sp. ciceris isolates based on polymorphic dart markers. cluster i represented 49% isolates from sz and 34% from nwpz and nepz. in the second cluster, 58% isolates were from nwpz and nhz and 34% from cz. the third cluster was smallest with the isolates mostly from nwpz and nepz and 38% from cz. fourth cluster represented 67% isolates from sz and remaining from cz and nwpz. few isolates from ap grouped separately and clustered at the top of the dendrogram. also few isolates were found to be isolated from major groups for instance isolates from br, hp, gj and mp.the pcoa based on polymorphic dart marker data derived from isolates, confirmed the four different groups found by upgma dendrogram. the two dimensions of pcoa explained % and % of the variation present in the genetic distance calculated between isolates. these two components were responsible for a total % of the variation observed among the isolates. the right-hand groups proved to be clustered around the pcoa x- axis with isolates originating them different agro-ecological zone. this group corresponded to the isolates from cluster iii of the upgma dendrogram. the left hand groups were spread widely along both the pcoa x- axis and pcoa y- axis. these groups represented the isolates from cluster-i, ii and iv of the upgma dendrogram.figure distribution of 110 f. oxysporum f. sp. ciceris isolates in different quadrants based principal coordinate analysis using dart markers. the principal coordinate i explain % and principal coordinate ii explain % variation. population structure of foc in order to assess the population structure and number of sub-populations, polymorphic dart markers were used on foc isolates employing structure. based on the maximum likelihood and delta k values, four sub-populations were determined in foc isolates. each vertical bar in the figure represents a single foc isolate and its inferred proportion of genetic admixture. the parts with different colours represent four different sub-populations. using a membership probability threshold of, isolates were assigned to sub-population isolate to sub-population isolates to sub-population and isolates to sub-population detailed information of sub-populations and respective isolate within each sub-population is provided in additional file sub-population and have genetic admixture, however sub-population have homogeneous genetic background.figure population structure among 110 f. oxysporum f. sp. ciceris isolates the highest delta k value is observed for k value indicating sub-populations among the foc isolates used in the study genetic composition of different sub-populations. allele frequency the allelic composition revealed the predominance of common and most frequent alleles in the total polymorphic alleles. foc population contained a lower proportion of rare alleles. among polymorphic markers, most frequent alleles were represented by % and common alleles contributed to %. however, unique alleles were detected in the foc isolates whose reaction did not match with any specific races. further, race specific unique alleles were also identified for different race specific isolates for instance, and. discussion the importance of understanding genetic diversity in any fungal species collection is critical for their effective management. although tremendous progress has been made in terms of availability of molecular markers to study the genetic diversity in foc; availability of sound, reliable and cost-effective marker platform is still lacking. in the present study, we present the first complete report on the development of dart marker platform for any fungal pathogen. to the best of our knowledge, this is the first attempt to develop dart markers for foc and investigation on the utility of these markers in genetic diversity of large number of foc isolates collected from various regions in india. we further demonstrated the pathogenic races, as determined by the disease reaction on differential cultivars-correlated to inferred dart genotyping. the dart technology combines a reduction of genome complexity with high throughput and cost-effective hybridization based polymorphism detection. the advantage of using these dart markers is that they can simultaneously type several thousand loci in a single array and provide a cost-effective and sequence independent tool for whole genome fingerprinting. the dart technology has been applied to a number of plants and animals. however, no information is available on any plant pathogen till date. in this study, the new dart platform for foc was found acceptable and provided robust information about the genetic diversity in this collection. out of dart markers, markers were found to be useful in providing a complete picture of genetic diversity in the foc collection of isolates. overall, the average pic value for dart marker was found to be lower than that observed in other plant species, where similar markers were developed, but comparable to that observed in pigeonpea and sugar beet. nearly nine per cent of dart markers have a pic value in the range of - and these markers therefore may be considered informative. dart markers developed in this study effectively detected diversity among foc isolates. all the three types of dart diversity analysis indicated that the different isolates were successfully classified by the marker system and reflected high genetic diversity. the phylogeny of the foc isolates inferred from dart markers by cluster analysis indicated major clusters among them. the subgroup clustering pattern with in each major cluster was consistent, indicating that the dart markers faithfully detected small differences among isolates. a similar finding was reported earlier from different markers systems like rapd, aflp and ssr with considerably less resolution and less transferability. virulence analysis on a set of differential cultivars is required to authenticate the groups generated by the molecular markers. in this study, virulence analysis based on standard set of differentials showed the existence of highly variable populations of foc in different agro-climatic zones in india. the reactions on differential cultivars of chickpea indicated the existence of races and in india. in addition few isolates did not show match with any of the race reaction and was grouped separately in both the dendrograms. previous studies based on differentials reported the presence of eight pathogenic races in foc based on disease reactions on a set of differential chickpea cultivars. race represented 47% of total foc isolates, of which most of the isolates grouped in cluster i. previous studies reported race only from ap and ka states of india, however present diversity based on dart indicated its presence in all the agro-ecological zones of india, with predominance in sz. the high genetic diversity among the race isolates of foc revealed its diverse geographical inhabitation under the present scenario. this widespread distribution of race can be attributed to the seed transmission through large exchange of germplasm that took place more in last one decade. among all the races, race is more widespread and has been reported in india, california, and the mediterranean region. race represented 29% of total foc isolates, of which most of the isolates were grouped into cluster ii. the race was previously confined to mediterranean basin. it was first reported from california later reported from spain, israel and morocco. occurrence of this new race of foc in india mainly appeared to be a recent introduction through seed transmission and pathogen genetic variability from accumulation of mutations over the time. according to jimenez et al., there may have little or no selection for resistance –breaking races of foc, minimizing the number of parallel events in acquisition of virulence, which are frequently observed in other system. thus, unlike other pathosystems there may have been little or no selection for resistance breaking races of foc, which minimizes the probabilities of obtaining parallel changes in virulence. they further stated that it is not probable that the stepwise evolution of races in foc resulted from the selection by specific resistance in chickpea populations, as widespread use of race a-resistant cultivars in india has not yet led to reports on development of race on the contrary, we speculate that stepwise evolution of races in foc resulted from selection by specific resistance in chickpea populations or repeated migration has occurred rather than the independent evolution of races in different regions or as expected from an ancient lineage race could be the common ancestor of all races. presence of race in india is also indicated by dubey et al.. further, there are reports that races and have similar virulence pattern on chickpea differentials and high genetic similarities. earlier researchers indicated that it is possible that misclassification of these isolates occurred during pathogenicity test. it is also possible that these two pathogenic races were not yet evolutionarily separated from each other to have developed diagnostic and separate pathogenicity test. the isolates grouped in cluster iii represent those isolates which had susceptible reaction with less virulence on jg cultivar and resistance reaction on all the other differential cultivars. this group corresponded to 19% of total foc isolates representing nz. difference in virulence among foc isolates originating from southern and northern partes of india has also been reported by dubey et al.. the distribution of virulence within and among populations of foc is probably the result of different selection pressure exerted by a specific resistant gene in the chickpea varieties cultivated in the area. this is in agreement with the observations by lebeda and petrzelova in the case of bremia lactucae. also, this could be the result of shift in the cultivation of chickpea from northern india to southern india, which further supported the low frequency of race originally reported from kanpur, north india. race disease reaction was not found in any of the isolates. the absence of race in the present collection is in agreement with gurjar et al.. they found completely different reaction of race and reported its resemblance to fusarium proliferatum based on phylogenetic analysis. the isolates in cluster iv mainly represent multiple race reaction race race race and race regardless of the origin, the similarities of race composition between the different zones of india indicated that repeated migration of infected seeds. the unique, common and most frequent alleles among the total polymorphic markers reported in the present study are novel features. such features, to the best of our knowledge, have not been reported in other diversity study. race specific alleles detected in the present study will be very useful for molecular profiling of a particular race. further, occurrence of common alleles will be useful for understanding the similarity and also molecular profiling of foc isolates from diverse geographical locations. two rare alleles observed specific to up location could be useful in the detection of location specific foc isolates.  the present study generated significant information in terms of pathogenic and genetic diversity of foc which could be used further for deployment of region-specific resistant varieties of chickpea. the study clearly highlighted the occurrence of multiple/new races of foc in india and suggests the continuous monitoring of changes in foc population to check the susceptibility of widely grown resistant cultivars against these races. the dart markers developed in the present study are powerful diagnostic tools for understanding the diversity in foc population in chickpea. in this study, we reported the successful development of dart marker platform for foc and their utility in genotyping of foc collections. this dart marker system complemented the microsatellite markers developed previously for foc. the high number of dart markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of variability in the foc population present in india, as well as provided support to know the changing race scenario in foc population. further, the dart markers identified in this study could be used to study the distribution of these foc races, and to facilitate the efficient deployment of available host resistance. these markers should assist in the early detection of introduced race, as well as of changes in the relative frequencies of different races that might occur in response to the use of resistant chickpea cultivars. the dart arrays developed for foc in this study are available with the center of excellence in genomics at icrisat. also the foc isolates used in the present study are available with legumes pathology unit at icrisat for further use by any scientific community. 
in this study, we developed dart markers for foc population, analysed the genetic diversity existing within and among foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of foc in india. diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. the advantages of using molecular markers to understand the distribution of genetic diversity in foc populations is well understood. the present study generated significant information in terms of pathogenic and genetic diversity of foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. ciceris, the causal agent of fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. the severity and yield losses of fusarium wilt differ from place to place owing to existence of physiological races among isolates. chickpeadartfusarium wiltmolecular markersracesvirulenceissue-copyright-statement© the author 2014 the high number of dart markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the foc population present in india, as well as provided support to know the changing race scenario in foc population. the recent development of diversity arrays technology offers new possibilities to study the diversity in pathogen population. diversity in foc population was compared with the phenotypic variability and it was found that dart markers were able to group the isolates consistent with its virulence group. a number of race-specific unique and rare alleles were also detected. the dart arrays revealed a total polymorphic markers with an average genotyping call rate of % and a scoring reproducibility of 100%. we report the successful development of dart markers for foc and their utility in genotyping of foc collections representing five chickpea growing agro-ecological zones of india. the dart markers were proved to be a powerful diagnostic tool to study the genotypic diversity in foc. cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of foc were partially classified based on geographical source.
 fusarium oxysporum f. sp. ciceris, the causal agent of fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. the severity and yield losses of fusarium wilt differ from place to place owing to existence of physiological races among isolates. diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. the advantages of using molecular markers to understand the distribution of genetic diversity in foc populations is well understood. the recent development of diversity arrays technology offers new possibilities to study the diversity in pathogen population. in this study, we developed dart markers for foc population, analysed the genetic diversity existing within and among foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of foc in india.  we report the successful development of dart markers for foc and their utility in genotyping of foc collections representing five chickpea growing agro-ecological zones of india. the dart arrays revealed a total polymorphic markers with an average genotyping call rate of % and a scoring reproducibility of 100%. cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of foc were partially classified based on geographical source. diversity in foc population was compared with the phenotypic variability and it was found that dart markers were able to group the isolates consistent with its virulence group. a number of race-specific unique and rare alleles were also detected.  the present study generated significant information in terms of pathogenic and genetic diversity of foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. the dart markers were proved to be a powerful diagnostic tool to study the genotypic diversity in foc. the high number of dart markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the foc population present in india, as well as provided support to know the changing race scenario in foc population. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chickpeadartfusarium wiltmolecular markersracesvirulenceissue-copyright-statement© the author 2014 