BACKGROUND
the small grass brachypodium distachyon  is fast emerging as a powerful model system to study questions unique to the grasses.
brachypodium possesses the physical and genomic attributes  necessary to be a modern model organism.
a truly tractable model grass is needed because the extremely powerful model dicot, arabidopsis thaliana, cannot be used to answer questions where dicot and grass biology diverge .
humans derive the majority of their food directly or indirectly from grasses and are projected to use increasing amounts of energy derived from grasses grown as biofuel crops.
thus, there is considerable need for a model grass to facilitate research aimed at improving grasses as grain, forage and energy crops to supply an ever increasing human population with food and energy.
the emergence of brachypodium as a model organism fulfills this need.
brachypodium is a typical grass in terms of architecture and growth habit  <cit> .
thus, brachypodium is an excellent functional model for all grasses including the large perennial grasses like switchgrass and miscanthus that are being developed as dedicated energy crops.
in this context, the small size , compact genome , and rapid generation time  of brachypodium will facilitate the application of modern high-throughput genomic technologies toward questions relevant to energy crops.
it is particularly important to have a model for emerging energy crops because their large size, large genomes and outcrossing nature make the use of many powerful experimental approaches  difficult in these species.
thus, it is not surprising that the rapid development of numerous tools for brachypodium is coincident with increased interest in research topics relevant to the development of grasses as biomass crops for the sustainable production of biofuels.
one important area of investigation is the unique cell wall of grasses.
in this context, brachypodium is an excellent system because its cell wall is typical of grasses including switchgrass and miscanthus  <cit> .
thus, knowledge in this area gained from brachypodium will be applicable to energy crops.
brachypodium is closely related to wheat and the small grains  <cit>  and thus, in addition to serving as a functional genomic model, holds promise as a structural genomic tool to aid the exploration of the huge genomes found in these crops.
several studies have already used the closely related species brachypodium sylvaticum as a tool to aid cloning projects in wheat and barley  <cit> .
in addition to serving as a traditional model, brachypodium is an excellent test-bed for transgenic approaches in the grasses because of an extremely efficient agrobacterium-mediated transformation system and a rapid generation time.
we currently obtain average transformation efficiencies over 50% for high-throughput production of t-dna tagged lines  and efficiencies ranging from 30–80% have been reported by three groups  <cit> .
.
other experimental resources that have been developed or are under development to enable brachypodium to be used as a model organism include: identification of growth conditions that allow rapid generation times , bac libraries, physical maps, methods for ethane methyl sulfonate and fast neutron mutagenesis, cdna libraries, and est sequences  <cit> .
in addition, a high-density genetic linkage map has been constructed  and, most importantly, is the imminent completion of the whole genome sequence including a large est sequencing component to aid annotation.
missing from the list of brachypodium resources is a large collection of diverse inbred lines and genetic markers.
there are currently only six freely available inbred diploid lines  <cit> .
some additional collections have been made, but are available only under a restrictive material transfer agreement  <cit> .
a diverse collection of well described, freely available inbred lines is essential to allow brachypodium to be used to study natural variation, and to allow positional cloning of induced mutations.
genetic markers are essential for many experiments including positional cloning, mapping quantitative trait loci, association mapping, ecotilling and analysis of genotypic diversity in populations.
pcr-based markers are particularly useful because they are fast, easy to score and can be used by any lab with standard molecular biology tools.
simple sequence repeats , also known as microsatellites, are genomic areas with simple short repeat units.
the number of repeats in these regions is highly polymorphic and thus markers developed from ssrs are particularly powerful.
both markers and a diverse collection of inbred lines are necessary for brachypodium to reach its full potential as a model system.
we addressed these needs by developing ssr markers and creating inbred lines from a diverse collection of brachypodium accessions collected from  <dig> locations throughout turkey.
RESULTS
ssr identification and initial survey of polymorphism
in total,  <dig>  ssrs were identified  using cutoff values of six repeats for dinucleotide repeats, five repeats for trinucleotide repeats, and four repeats for tetranucleotide repeats.
the most common repeat from the ests was the trinucleotide repeat ccg which comprised 36% of all est-derived ssrs and the most common repeat from the bes was the dinucleotide repeat ga which comprised 40% of all bes-derived repeats .
the higher percentage of trinucleotide repeats in the ests suggests that many est ssrs are constrained by being in coding regions.
to identify robust, polymorphic markers for population studies we determined the level of ssr polymorphism in a small collection of six brachypodium lines  using  <dig> primer pairs  .
out of the  <dig> primer pairs tested,  <dig>  produced strong bands.
of those,  <dig>  primer pairs identified polymorphisms between at least two of the six lines.
as has been observed in other systems, ssrs with more repeats were more polymorphic than ssrs with fewer repeats.
for example, ssrs with five repeat units had an average of  <dig>  alleles whereas ssrs with  <dig> or more repeats had  <dig>  alleles.
the diversity of ssr alleles found in this initial population indicates that there is sufficient ssr diversity to use ssr markers for mapping or population analysis.
full details of product sizes for all markers are presented in additional file  <dig>
collection of new accessions and creation of inbred lines
two new collections of brachypodium accessions were used in this study.
the first collection was made by mt between june  <dig> and july  <dig>   <dig> from eight locations in turkey .
for three locations  an area of ~ <dig>  m <dig> was sampled by collecting seed from individual plants scattered across the site.
bulk collections of several plants at different sub-sites were also made.
inbred lines were made from each of the different individual plants that were collected.
from five sites  only one bulk collection from plants growing in small area of a few square meters was made.
for these locations eight inbred lines were made by sub-sampling the bulk seed.
all sites except tekirdag were overgrazed hilly pasture with continental climate .
tekirdag lies near the marmara sea and so has a more maritime climate .
the tekirdag site is also forested and so was shaded and not subject to grazing.
the  <dig> inbred lines developed from material collected by mt were named using the first three letters of a nearby town as the prefix.
the locations and other details of these collections are summarized in additional file  <dig>  these lines were inbred for two generations.
the second collection was made independently by hb in  <dig> from  <dig> locations across turkey .
full details of these collections, the development of inbred lines and phenotypic analysis will be published elsewhere.
one hundred twenty one inbred diploid lines from this collection were used in the present study .
these inbred lines were placed into  <dig> phenotypic groups and the naming convention consists of the group prefix  followed by a unique letter for each inbred line.
the phenotypic groups bdtr <dig> and bdtr <dig> were polyploid and were not included in the present study.
for this collection it is important to note that lines with the same prefix  do not necessarily come from the same location .
in addition to the new collections, six previously described inbred lines, bd1- <dig>  bd2- <dig>  bd3- <dig>  bd18- <dig>  bd <dig> and bd21- <dig> were also used  <cit> .
phenotypic characterization
for the inbred lines developed from material collected by mt, we examined flowering time, vernalization requirements, seed size and presence of hairs on the lemma.
this phenotypic analysis focused on easily scored phenotypes in order to determine if there was major phenotypic diversity in the collection.
it is not intended to be an exhaustive examination of phenotype by environment interaction but rather to provide a resource to allow users to select lines with potentially useful variation in traits of interest.
flowering time was highly dependent upon the length of vernalization .
all the lines required longer vernalization times to induce flowering than bd <dig>  bd21- <dig>  bd2- <dig> or bd3- <dig>  four or five weeks of vernalization was sufficient to induce rapid flowering in all lines except those from tekirdag.
the lines from tekirdag required much longer vernalization times from 8– <dig> weeks.
growth under long day conditions  has previously been shown to promote the flowering of bd <dig>  bd21- <dig>  bd2- <dig> and bd3- <dig> in the absence of vernalization  <cit> .
however, these long day conditions failed to induce rapid flowering of any of the material collected by mt or hb.
seed size ranged from  <dig>  to  <dig>  mg/seed for the diploid lines.
the average seed size was  <dig>  mg/seed .
there were significant differences between the average seed sizes from the different populations .
most of this difference was due to the smaller seed size of the lines from tekirdag.
the average seed size of the lines from tekirdag  was significantly  less than the average seeds size of the lines from all other locations .
the lines from tekirdag had short hairs on their lemmas and appeared smooth whereas most other lines had very hairy lemmas .
bd18- <dig> seeds had short hairs similar to the tek lines.
bd18- <dig> also requires a long vernalization similar to the tek lines.
one line, koz- <dig> had no visible hairs on the lemma .
we also observed variation in the number of inflorescence branches, number of seeds per branch, and the angle of the seeds and branches .
it should be noted that the number of branches produced by a given line is highly dependent upon vernalization.
therefore, only lines grown under the same conditions and vernalized such that they flower at the same size can be compared.
longer vernalization times lead to fewer branches, fewer tillers and lower seed production.
ploidy was determined by flow cytometry  <cit> .
for the material collected by mt, c-values from representatives of each population were determined first.
later, representative inbred lines were tested to confirm initial results.
five locations contained only diploid accessions, two locations had only polyploid accessions and one location had both diploid and polyploid plants .
one inbred line, adi-p <dig>  appeared phenotypically polyploid, but was collected from a location where c-values from population samples indicated diploidy.
flow cytometry of adi-p <dig> confirmed that it was indeed polyploid while other inbred lines from adiyaman were diploid .
* the value presented is the average of 4– <dig> c-values obtained from individual plants.
while ploidy level in some grass species  cannot be distinguished visually  <cit> , in brachypodium when grown side by side polyploid lines were easily distinguished from diploid lines by visual examination .
two forms of polyploids were apparent.
one form typified by adi-p <dig>  lines from balli, and bdtr <dig> lines has much larger seeds.
for example, adi-p <dig> has an average seed size of  <dig>  mg, much larger than the  <dig>  mg/seed average for all diploid lines .
this form also had much thicker and hairier stems .
the other form typified by lines from iskenderun and bdtr <dig> lines had seeds similar in size to diploid seeds, but with a more pronounced crease .
these polyploid lines also tended to bear more seeds per spike, though this trait is variable depending upon the conditions.
another difference between polyploid and diploid lines is the degree to which anthers exert.
anthers from flowers of both polyploid forms often exert whereas, under our conditions, anthers rarely exert from diploid flowers.
ssr analysis and inbreeding
based on our initial survey of ssr sizes in six inbred bd_lines, we selected  <dig> ssr markers that produced robust bands and were highly polymorphic among the bd_collection to genotype the entire collection .
the number of alleles per marker ranged from three to  <dig> to and the average number of alleles was  <dig>  .
summary statistics for the markers are presented in additional file  <dig> and all the ssr sizes are presented in additional file  <dig>  out of the  <dig>  genotypes determined, only four heterozygous plants that were each heterozygous for one marker were found.
this indicates that the lines are highly inbred.
to determine the prevalence of brachypodium self pollination in the wild, for  <dig> of the lines we genotyped the first plant to be grown under greenhouse conditions.
since these plants had undergone no inbreeding in the lab they are representative of wild plants.
these 'wild' plants were overwhelmingly homozygous.
in fact, only one marker from one plant was heterozygous.
there was an average of  <dig>  alleles per marker per population in this sample.
therefore, if there was a significant amount of outcrossing, we would have seen many more heterozygous individuals.
this indicates that brachypodium is primarily a selfing species under field conditions.
in further support of brachypodium's inbreeding nature, we have observed that diploid flowers rarely open under our greenhouse and growth chamber conditions.
under certain environmental conditions  we have observed open flowers on diploid plants grown outside.
however, inspection of these open flowers revealed that the anthers had already dehisced on the stigmas under the fold of the palea.
thus, even open flowers are expected to produce an overwhelming proportion of self pollinations.
due to the high degree of homozygosity we stopped inbreeding the lines developed from mt collections after only two single seed descent generations under laboratory conditions.
* pcr product sizes for all lines can be found in additional file  <dig>
to determine the rate of outcrossing under laboratory conditions, we monitored the rate at which pollen from transgenic plants containing a constitutively expressed gus reporter gene pollinated wild-type plants.
to maximize the chance for cross-pollination, we surrounded single wild-type plants with 10– <dig> transgenic plants in a single  <dig> cm pot and tied the plants together as they grew in order to bring the flowers into close proximity.
to determine how often transgenic pollen fertilized non-transgenic ovules, we scored the progeny of the wild-type plants for expression of the gus reporter gene.
out of  <dig>  progeny  from  <dig> wild-type plants none expressed gus.
this indicates that the rate of outcrossing under 'worst case' laboratory conditions is exceedingly small.
to determine the relationship between inbred lines we constructed a consensus neighbor-joining tree based on  <dig> shared allele bootstrap trees.
an examination of the tree reveals two main groups with high bootstrap support .
one group consists of lines from tekirdag, bd1- <dig> and the bdtr <dig> and bdtr <dig> groups.
these lines are also naturally grouped by their smaller seeds, longer vernalization requirements and nearly hairless lemmas .
the other group consists of all the other lines from turkey and iraq.
this large group branches into smaller groups, however there is little bootstrap support for the larger branches.
there are, however, smaller subgroups with very high bootstrap support.
one striking feature is that the  <dig> bdtr groupings form very tight clusters despite being collected from different locations.
conversely, lines collected from adiyaman, gaziantep and kahta fall into several different groups.
in addition to the shared allele tree presented, we constructed several other trees using the other functions in powermarker.
in all cases, the two main groups and small subgroups with good bootstrap support were apparent .
discussion
the successful implementation of brachypodium as a model for the study of natural diversity and the positional cloning of induced mutations is dependent upon the phenotypic and genotypic diversity found within the species.
brachypodium occupies a variety of habitats including hot interior regions, cooler coastal areas and colder mountainous regions suggesting the existence of considerable genetic diversity.
the natural range of brachypodium is centered around the mediterranean extending north into europe and south into the indian subcontinent  <cit> .
within this region, turkey is expected to be a rich source of brachypodium diversity because it covers all the habitats mentioned above.
therefore, we have sampled this region heavily .
our results clearly indicate that there is sufficient phenotypic and genotypic diversity within this brachypodium collection to facilitate studies of natural diversity and allow efficient positional cloning of induced mutations.
we had no difficulties identifying polymorphic ssr markers and noted significant differences in easily scored phenotypes like seed size, vernalization requirements, inflorescence architecture and the presence of hairs.
this agrees with other groups who have noted diversity in various traits including disease resistance  <cit> .
the extremely high degree of homozygosity observed in wild brachypodium plants indicates that brachypodium is primarily a selfing species.
this is not surprising because as we and others have observed, pollination typically occurs in a closed flower  <cit> .
from a practical perspective, inbreeding simplifies the maintenance of pure lines under laboratory conditions.
indeed, our analysis of pollen flow suggests that even under a 'worst case' scenario it is easy to maintain pure lines.
it also means that wild collections do not need to be inbred for many generations to achieve a high degree of homozygosity.
the consensus tree based on ssr polymorphisms clearly shows two major groups with very high bootstrap support .
the smaller group containing the tek lines shared several phenotypic traits including: long vernalization requirements, seed size, and near absence of hairs on the lemmas.
the larger group contained lines from turkey and iraq.
within this group were tight clusters that contained all the lines from the different bdtr groups that were previously grouped together based on phenotypic similarity.
thus, there was remarkable correlation between the phenotypic groupings and the ssr profiles.
two lines of particular interest, bd <dig> the line whose genome was sequenced and bd21- <dig> a line that is efficiently transformed were found to be more closely related to one another than to any other line, but they are clearly distinct.
the diversity of homozygous genotypes found within very small areas suggests that considerable long-distance seed dispersal takes place.
this is consistent with the presence of hairs on the firmly attached lemmas of most lines.
these hairs aid seed dispersal by animals and humans.
the lack of bootstrap support for the major branches in the largest group suggests that, despite being primarily inbreeding, there is substantial genetic exchange over longer timescales.
CONCLUSIONS
in this study we have demonstrated that considerable genotypic and phenotypic variation exists within this brachypodium collection.
this diversity will allow scientific methods that exploit natural diversity to be applied to brachypodium.
the geographic distribution of ssr genotypes suggests that long-distance seed dispersal plays a significant role in the population structure of brachypodium.
the ssr markers and inbred lines developed in this study are a significant contribution to the brachypodium resources already available and are freely available to the scientific community.

 the small grass brachypodium distachyon is fast emerging as a powerful model system to study questions unique to the grasses. brachypodium possesses the physical and genomic attributes necessary to be a modern model organism. a truly tractable model grass is needed because the extremely powerful model dicot, arabidopsis thaliana, cannot be used to answer questions where dicot and grass biology diverge. humans derive the majority of their food directly or indirectly from grasses and are projected to use increasing amounts of energy derived from grasses grown as biofuel crops. thus, there is considerable need for a model grass to facilitate research aimed at improving grasses as grain, forage and energy crops to supply an ever increasing human population with food and energy. the emergence of brachypodium as a model organism fulfills this need. brachypodium is a typical grass in terms of architecture and growth habit. thus, brachypodium is an excellent functional model for all grasses including the large perennial grasses like switchgrass and miscanthus that are being developed as dedicated energy crops. in this context, the small size, compact genome, and rapid generation time of brachypodium will facilitate the application of modern high-throughput genomic technologies toward questions relevant to energy crops. it is particularly important to have a model for emerging energy crops because their large size, large genomes and outcrossing nature make the use of many powerful experimental approaches difficult in these species. thus, it is not surprising that the rapid development of numerous tools for brachypodium is coincident with increased interest in research topics relevant to the development of grasses as biomass crops for the sustainable production of biofuels. one important area of investigation is the unique cell wall of grasses. in this context, brachypodium is an excellent system because its cell wall is typical of grasses including switchgrass and miscanthus. thus, knowledge in this area gained from brachypodium will be applicable to energy crops. brachypodium is closely related to wheat and the small grains and thus, in addition to serving as a functional genomic model, holds promise as a structural genomic tool to aid the exploration of the huge genomes found in these crops. several studies have already used the closely related species brachypodium sylvaticum as a tool to aid cloning projects in wheat and barley. in addition to serving as a traditional model, brachypodium is an excellent test-bed for transgenic approaches in the grasses because of an extremely efficient agrobacterium-mediated transformation system and a rapid generation time. we currently obtain average transformation efficiencies over 50% for high-throughput production of t-dna tagged lines and efficiencies ranging from 30–80% have been reported by three groups.. other experimental resources that have been developed or are under development to enable brachypodium to be used as a model organism include: identification of growth conditions that allow rapid generation times, bac libraries, physical maps, methods for ethane methyl sulfonate and fast neutron mutagenesis, cdna libraries, and est sequences. in addition, a high-density genetic linkage map has been constructed and, most importantly, is the imminent completion of the whole genome sequence including a large est sequencing component to aid annotation. missing from the list of brachypodium resources is a large collection of diverse inbred lines and genetic markers. there are currently only six freely available inbred diploid lines. some additional collections have been made, but are available only under a restrictive material transfer agreement. a diverse collection of well described, freely available inbred lines is essential to allow brachypodium to be used to study natural variation, and to allow positional cloning of induced mutations. genetic markers are essential for many experiments including positional cloning, mapping quantitative trait loci, association mapping, ecotilling and analysis of genotypic diversity in populations. pcr-based markers are particularly useful because they are fast, easy to score and can be used by any lab with standard molecular biology tools. simple sequence repeats, also known as microsatellites, are genomic areas with simple short repeat units. the number of repeats in these regions is highly polymorphic and thus markers developed from ssrs are particularly powerful. both markers and a diverse collection of inbred lines are necessary for brachypodium to reach its full potential as a model system. we addressed these needs by developing ssr markers and creating inbred lines from a diverse collection of brachypodium accessions collected from locations throughout turkey.  ssr identification and initial survey of polymorphism in total, ssrs were identified using cutoff values of six repeats for dinucleotide repeats, five repeats for trinucleotide repeats, and four repeats for tetranucleotide repeats. the most common repeat from the ests was the trinucleotide repeat ccg which comprised 36% of all est-derived ssrs and the most common repeat from the bes was the dinucleotide repeat ga which comprised 40% of all bes-derived repeats. the higher percentage of trinucleotide repeats in the ests suggests that many est ssrs are constrained by being in coding regions. to identify robust, polymorphic markers for population studies we determined the level of ssr polymorphism in a small collection of six brachypodium lines using primer pairs. out of the primer pairs tested, produced strong bands. of those, primer pairs identified polymorphisms between at least two of the six lines. as has been observed in other systems, ssrs with more repeats were more polymorphic than ssrs with fewer repeats. for example, ssrs with five repeat units had an average of alleles whereas ssrs with or more repeats had alleles. the diversity of ssr alleles found in this initial population indicates that there is sufficient ssr diversity to use ssr markers for mapping or population analysis. full details of product sizes for all markers are presented in additional file collection of new accessions and creation of inbred lines two new collections of brachypodium accessions were used in this study. the first collection was made by mt between june and july from eight locations in turkey. for three locations an area of ~ m was sampled by collecting seed from individual plants scattered across the site. bulk collections of several plants at different sub-sites were also made. inbred lines were made from each of the different individual plants that were collected. from five sites only one bulk collection from plants growing in small area of a few square meters was made. for these locations eight inbred lines were made by sub-sampling the bulk seed. all sites except tekirdag were overgrazed hilly pasture with continental climate. tekirdag lies near the marmara sea and so has a more maritime climate. the tekirdag site is also forested and so was shaded and not subject to grazing. the inbred lines developed from material collected by mt were named using the first three letters of a nearby town as the prefix. the locations and other details of these collections are summarized in additional file these lines were inbred for two generations. the second collection was made independently by hb in from locations across turkey. full details of these collections, the development of inbred lines and phenotypic analysis will be published elsewhere. one hundred twenty one inbred diploid lines from this collection were used in the present study. these inbred lines were placed into phenotypic groups and the naming convention consists of the group prefix followed by a unique letter for each inbred line. the phenotypic groups bdtr and bdtr were polyploid and were not included in the present study. for this collection it is important to note that lines with the same prefix do not necessarily come from the same location. in addition to the new collections, six previously described inbred lines, bd1- bd2- bd3- bd18- bd and bd21- were also used. phenotypic characterization for the inbred lines developed from material collected by mt, we examined flowering time, vernalization requirements, seed size and presence of hairs on the lemma. this phenotypic analysis focused on easily scored phenotypes in order to determine if there was major phenotypic diversity in the collection. it is not intended to be an exhaustive examination of phenotype by environment interaction but rather to provide a resource to allow users to select lines with potentially useful variation in traits of interest. flowering time was highly dependent upon the length of vernalization. all the lines required longer vernalization times to induce flowering than bd bd21- bd2- or bd3- four or five weeks of vernalization was sufficient to induce rapid flowering in all lines except those from tekirdag. the lines from tekirdag required much longer vernalization times from 8– weeks. growth under long day conditions has previously been shown to promote the flowering of bd bd21- bd2- and bd3- in the absence of vernalization. however, these long day conditions failed to induce rapid flowering of any of the material collected by mt or hb. seed size ranged from to mg/seed for the diploid lines. the average seed size was mg/seed. there were significant differences between the average seed sizes from the different populations. most of this difference was due to the smaller seed size of the lines from tekirdag. the average seed size of the lines from tekirdag was significantly less than the average seeds size of the lines from all other locations. the lines from tekirdag had short hairs on their lemmas and appeared smooth whereas most other lines had very hairy lemmas. bd18- seeds had short hairs similar to the tek lines. bd18- also requires a long vernalization similar to the tek lines. one line, koz- had no visible hairs on the lemma. we also observed variation in the number of inflorescence branches, number of seeds per branch, and the angle of the seeds and branches. it should be noted that the number of branches produced by a given line is highly dependent upon vernalization. therefore, only lines grown under the same conditions and vernalized such that they flower at the same size can be compared. longer vernalization times lead to fewer branches, fewer tillers and lower seed production. ploidy was determined by flow cytometry. for the material collected by mt, c-values from representatives of each population were determined first. later, representative inbred lines were tested to confirm initial results. five locations contained only diploid accessions, two locations had only polyploid accessions and one location had both diploid and polyploid plants. one inbred line, adi-p appeared phenotypically polyploid, but was collected from a location where c-values from population samples indicated diploidy. flow cytometry of adi-p confirmed that it was indeed polyploid while other inbred lines from adiyaman were diploid. * the value presented is the average of 4– c-values obtained from individual plants. while ploidy level in some grass species cannot be distinguished visually, in brachypodium when grown side by side polyploid lines were easily distinguished from diploid lines by visual examination. two forms of polyploids were apparent. one form typified by adi-p lines from balli, and bdtr lines has much larger seeds. for example, adi-p has an average seed size of mg, much larger than the mg/seed average for all diploid lines. this form also had much thicker and hairier stems. the other form typified by lines from iskenderun and bdtr lines had seeds similar in size to diploid seeds, but with a more pronounced crease. these polyploid lines also tended to bear more seeds per spike, though this trait is variable depending upon the conditions. another difference between polyploid and diploid lines is the degree to which anthers exert. anthers from flowers of both polyploid forms often exert whereas, under our conditions, anthers rarely exert from diploid flowers. ssr analysis and inbreeding based on our initial survey of ssr sizes in six inbred bd_lines, we selected ssr markers that produced robust bands and were highly polymorphic among the bd_collection to genotype the entire collection. the number of alleles per marker ranged from three to to and the average number of alleles was. summary statistics for the markers are presented in additional file and all the ssr sizes are presented in additional file out of the genotypes determined, only four heterozygous plants that were each heterozygous for one marker were found. this indicates that the lines are highly inbred. to determine the prevalence of brachypodium self pollination in the wild, for of the lines we genotyped the first plant to be grown under greenhouse conditions. since these plants had undergone no inbreeding in the lab they are representative of wild plants. these'wild' plants were overwhelmingly homozygous. in fact, only one marker from one plant was heterozygous. there was an average of alleles per marker per population in this sample. therefore, if there was a significant amount of outcrossing, we would have seen many more heterozygous individuals. this indicates that brachypodium is primarily a selfing species under field conditions. in further support of brachypodium's inbreeding nature, we have observed that diploid flowers rarely open under our greenhouse and growth chamber conditions. under certain environmental conditions we have observed open flowers on diploid plants grown outside. however, inspection of these open flowers revealed that the anthers had already dehisced on the stigmas under the fold of the palea. thus, even open flowers are expected to produce an overwhelming proportion of self pollinations. due to the high degree of homozygosity we stopped inbreeding the lines developed from mt collections after only two single seed descent generations under laboratory conditions. * pcr product sizes for all lines can be found in additional file to determine the rate of outcrossing under laboratory conditions, we monitored the rate at which pollen from transgenic plants containing a constitutively expressed gus reporter gene pollinated wild-type plants. to maximize the chance for cross-pollination, we surrounded single wild-type plants with 10– transgenic plants in a single cm pot and tied the plants together as they grew in order to bring the flowers into close proximity. to determine how often transgenic pollen fertilized non-transgenic ovules, we scored the progeny of the wild-type plants for expression of the gus reporter gene. out of progeny from wild-type plants none expressed gus. this indicates that the rate of outcrossing under'worst case' laboratory conditions is exceedingly small. to determine the relationship between inbred lines we constructed a consensus neighbor-joining tree based on shared allele bootstrap trees. an examination of the tree reveals two main groups with high bootstrap support. one group consists of lines from tekirdag, bd1- and the bdtr and bdtr groups. these lines are also naturally grouped by their smaller seeds, longer vernalization requirements and nearly hairless lemmas. the other group consists of all the other lines from turkey and iraq. this large group branches into smaller groups, however there is little bootstrap support for the larger branches. there are, however, smaller subgroups with very high bootstrap support. one striking feature is that the bdtr groupings form very tight clusters despite being collected from different locations. conversely, lines collected from adiyaman, gaziantep and kahta fall into several different groups. in addition to the shared allele tree presented, we constructed several other trees using the other functions in powermarker. in all cases, the two main groups and small subgroups with good bootstrap support were apparent. discussion the successful implementation of brachypodium as a model for the study of natural diversity and the positional cloning of induced mutations is dependent upon the phenotypic and genotypic diversity found within the species. brachypodium occupies a variety of habitats including hot interior regions, cooler coastal areas and colder mountainous regions suggesting the existence of considerable genetic diversity. the natural range of brachypodium is centered around the mediterranean extending north into europe and south into the indian subcontinent. within this region, turkey is expected to be a rich source of brachypodium diversity because it covers all the habitats mentioned above. therefore, we have sampled this region heavily. our results clearly indicate that there is sufficient phenotypic and genotypic diversity within this brachypodium collection to facilitate studies of natural diversity and allow efficient positional cloning of induced mutations. we had no difficulties identifying polymorphic ssr markers and noted significant differences in easily scored phenotypes like seed size, vernalization requirements, inflorescence architecture and the presence of hairs. this agrees with other groups who have noted diversity in various traits including disease resistance. the extremely high degree of homozygosity observed in wild brachypodium plants indicates that brachypodium is primarily a selfing species. this is not surprising because as we and others have observed, pollination typically occurs in a closed flower. from a practical perspective, inbreeding simplifies the maintenance of pure lines under laboratory conditions. indeed, our analysis of pollen flow suggests that even under a'worst case' scenario it is easy to maintain pure lines. it also means that wild collections do not need to be inbred for many generations to achieve a high degree of homozygosity. the consensus tree based on ssr polymorphisms clearly shows two major groups with very high bootstrap support. the smaller group containing the tek lines shared several phenotypic traits including: long vernalization requirements, seed size, and near absence of hairs on the lemmas. the larger group contained lines from turkey and iraq. within this group were tight clusters that contained all the lines from the different bdtr groups that were previously grouped together based on phenotypic similarity. thus, there was remarkable correlation between the phenotypic groupings and the ssr profiles. two lines of particular interest, bd the line whose genome was sequenced and bd21- a line that is efficiently transformed were found to be more closely related to one another than to any other line, but they are clearly distinct. the diversity of homozygous genotypes found within very small areas suggests that considerable long-distance seed dispersal takes place. this is consistent with the presence of hairs on the firmly attached lemmas of most lines. these hairs aid seed dispersal by animals and humans. the lack of bootstrap support for the major branches in the largest group suggests that, despite being primarily inbreeding, there is substantial genetic exchange over longer timescales.  in this study we have demonstrated that considerable genotypic and phenotypic variation exists within this brachypodium collection. this diversity will allow scientific methods that exploit natural diversity to be applied to brachypodium. the geographic distribution of ssr genotypes suggests that long-distance seed dispersal plays a significant role in the population structure of brachypodium. the ssr markers and inbred lines developed in this study are a significant contribution to the brachypodium resources already available and are freely available to the scientific community. 
phenotypic analysis of this new germplasm resource revealed considerable variation in flowering time, seed size, and plant architecture. the inbreeding nature of brachypodium was confirmed by an extremely high level of homozygosity in wild plants and a lack of cross-pollination under laboratory conditions. to characterize the natural diversity of brachypodium and provide a valuable new tool to the growing list of resources available to brachypodium researchers, we created and characterized a large, diverse collection of inbred lines. the resources developed in this study are freely available to the research community and will facilitate experimental applications based on natural diversity. taken together, the inbreeding nature and genotypic diversity observed at individual locations suggest a significant amount of long-distance seed dispersal. brachypodium distachyon is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. an analysis of diploid lines from locations with ssr markers showed considerable genotypic diversity. there was some correlation between ssr genotypes and broad geographic regions, but there was also a high level of genotypic diversity at individual locations.
 brachypodium distachyon is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. to characterize the natural diversity of brachypodium and provide a valuable new tool to the growing list of resources available to brachypodium researchers, we created and characterized a large, diverse collection of inbred lines.  we developed inbred lines from eight locations in turkey. to enable genotypic characterization of this collection, we created ssr markers from bac end and est sequences. an analysis of diploid lines from locations with ssr markers showed considerable genotypic diversity. there was some correlation between ssr genotypes and broad geographic regions, but there was also a high level of genotypic diversity at individual locations. phenotypic analysis of this new germplasm resource revealed considerable variation in flowering time, seed size, and plant architecture. the inbreeding nature of brachypodium was confirmed by an extremely high level of homozygosity in wild plants and a lack of cross-pollination under laboratory conditions.  taken together, the inbreeding nature and genotypic diversity observed at individual locations suggest a significant amount of long-distance seed dispersal. the resources developed in this study are freely available to the research community and will facilitate experimental applications based on natural diversity. 