BACKGROUND
barley  is a diploid  economically important cereal crop and genetic model for small grain temperate cereals.
golden promise  is a two-rowed uk spring barley cultivar, and is currently the most responsive genotype for barley genetic transformation.
also, because of its unique properties, the malt extracted from gp is used to distil a number of signature single malt scotch whiskies such as macallan and glengoyne.
it is a primary induced gamma-ray mutant derivative of the barley cultivar maythorpe, and is known to contain a mutation in breviaristatum-e .
this mutation in ari-e in gp  causes a semi-dwarfing phenotype that has been used widely in barley cultivar development  to shorten straw length and reduce the severity of lodging.
gp is also susceptible to several fungal pathogens, has short awns , reduced internode length and shows a measure of tolerance to salt  <cit> .
genetic analysis has previously located ari-e.gp to barley chromosome 5h as a quantitative trait locus  influencing plant height, and physiological studies have confirmed its relative insensitivity to the addition of exogenous gibberellic acid   <cit> .
the ari-e gene has not yet been cloned although it was recently mapped as a height qtl using the tools of contemporary biometrical genetics in a complex three-way cross  <cit> .
over the past two decades, many molecular tools have been developed in barley to enable genetic research  <cit> .
the primary focus has been the construction of molecular marker-based genetic linkage maps that can be leveraged for mapping genes of interest and subsequent marker assisted selection in breeding programs.
these have been applied to discover, dissect and manipulate genes determining a range of simple and complex traits.
because of their value, accompanied by their increasing use in genetics and breeding, there has been a continual drive to both reduce marker costs and to avoid ascertainment issues  <cit>  while at the same time enhancing flexibility and marker throughput per assay.
it is therefore appropriate that new developments in marker technology are both explored and thoroughly evaluated against the current state of the art.
now that next generation sequencing  technology has been shown to be capable of discovering and genotyping thousands of markers across almost any genome of interest at low cost and in a single step, a current debate is whether sequence-based genotyping methods are ready to replace many of the established and widely used tools such as highly-multiplex single nucleotide polymorphism  platforms  <cit> .
available sequence-based genotyping methods generally rely upon the use of restriction enzymes to produce a reduced representation of the non-repetitive  regions of the genome.
restriction site-associated genomic dna  typing is such an approach and has been used in several species for the construction of linkage maps and application in qtl analyses  <cit> .
in barley, a rad linkage map was recently produced in a double haploid population and used for qtl analysis  <cit> .
elshire and colleagues  <cit>  subsequently described a similar but more straightforward method of genotyping by sequencing  which works effectively in 96-well  plate assays.
gbs was originally developed for high-resolution association studies in maize  <cit>  and, like rad, has been extended to a range of species with complex genomes.
a two-enzyme gbs protocol has now been developed that produces a uniform library for sequencing and has been applied to both wheat and barley  <cit> .
this gbs approach has been shown to be suited to genetic analysis of rapeseed, lupin, lettuce, switchgrass, soybean, and maize  <cit> .
in this report, our biological objective was to identify at high resolution the genetic location of the ari-e.gp semi-dwarfing gene of cultivated barley.
however, as a sequence assembly of the barley genome has just been published  <cit> , we also wanted to use a sequence-based genetic marker methodology that would in principle allow us to link directly to the genome sequence assemblies and physical map, ultimately as a shortcut to facilitate the identification of the ari-e gene.
we therefore chose to explore use of the two-enzyme based gbs method, using digestion of genomic dna with a six-base methylation sensitive ‘rare-cutter’ and a four-base ‘common cutter’ enzyme.
we combined this with the illumina ngs platform and developed a downstream informatics pipeline to discover co-dominant  in an f <dig> single-seed descent mapping population from a golden promise  by morex  cross.
in contrast to gp, mx is a tall spring six-rowed north american barley variety with desirable malting and brewing characteristics.
most importantly, mx is the reference cultivar used in the barley genome sequencing efforts.
our genetic analysis using gbs data from the recombinant inbred  population confirmed the location of ari-e.gp on barley chromosome 5h.
in the process we discovered  <dig>  high-confidence snps that we could associate with contigs in the ngs sequence assemblies and physical map.
RESULTS
golden promise by morex population  and variation in plant height
a recombinant inbred line  population of 160 f <dig> single-seed descent lines from a gp by mx cross was developed over seven years, from 2003– <dig>  at the james hutton institute.
the 136 f <dig> rils used in this study comprised  <dig> two-rowed and  <dig> six-rowed accessions.
the population segregates quantitatively for height as shown in figure  <dig> varying between different rils from  <dig> to 130 cm.
asymmetric transgressive segregation of plant height across the gpmx population can be observed – there were around  <dig> lines taller than morex, but only about  <dig> shorter than golden promise.
the pearson correlation between the heights in the two years was  <dig> .
two lines showed a marked difference in height between the two years.
generation of psti reference sequences from barley genome assemblies
to facilitate genetic analysis by gbs we first extracted a set of 64 bp reference sequences flanking all predicted psti restriction sites from barley genome assemblies of the cultivars morex   <cit> ), bowman  and barke  using the ‘restrict’ program from the emboss suite of tools .
for cultivar morex,  <dig>  restriction sites were identified, yielding a total of  <dig>  gbs reference sequences present on  <dig>  unique morex genome assembly contigs.
of all the identified sites,  <dig>  flanking sequences had to be excluded because the restriction site was too close to the start or end of an assembled genomic contig and therefore extraction of the full 64 bp sequence was impossible.
extraction of additional sequences unique to the genome assemblies of cultivars bowman and barke yielded a further  <dig>  and  <dig>  sequences, respectively.
after removal of chloroplast  sequences a total of  <dig>  reference sequences remained that were subsequently used for read mapping.
more than half  of the 64 bp reference sequences stemmed from morex contigs that contain regions of homology to full length cdnas or expressed genes, previously mapped genetic markers  or sequences that have chromosome arm assignments based on survey sequencing of flow sorted chromosome arms  <cit> .
gbs reads of gpmx
we generated three 48-plex gpmx gbs libraries  representing all  <dig> progenies and the parents, which were repeatedly represented in each library for qc purposes.
we used psti combined with msei to digest genomic dna, with the psti overhang sequence located in the barcode adapter adjacent to the barcode sequence, and the msei overhang sequence located in common y-adapter  <cit>  .
single-end sequencing starting from the barcoded adapter was performed using illumina chemistry.
pilot sequencing the gpmx_ <dig> library on an illumina gaii platform generated more than  <dig> million single-end reads of 72 bp in length.
of these, over 58 m reads were categorised as having a correct barcode and psti overhang sequence .
further sequencing of all three gpmx population libraries, each on one lane of an illumina hiseq <dig>  generated a total of 622 m reads of 100 bp and more than 482 m remaining as categorised reads .
the average number of categorised reads obtained per lane was  <dig>  m on the illumina ga ii and 160 m on the illumina hiseq <dig>  by applying various filtering criteria  in the reads), the percentages of categorised reads were  <dig> %  and  <dig> % .
after deconvolution the distribution of the number of reads per sample ranged from  <dig>  to  <dig> , <dig> .
the read distribution was relatively even across the population, with only  <dig> of the  <dig> lines having less than a million reads.
for the parents, we obtained  <dig>  m reads from golden promise and  <dig>  m from morex, due to repeat sequencing.
all sequence reads generated from gpmx were submitted to the sequence read archive section of the european nucleotide archive  .
co-dominant markers from the gpmx population datasets
in total, 461 m categorized reads from the gpmx mapping population were mapped to the 64 bp reference sequences using the bowtie mapping tool  <cit> .
in order to reduce the number of false positive snps during downstream analysis, only a single mismatch per read was allowed, and only uniquely mapped reads were included, leaving 46% of 461 m reads mapped to the reference.
these categorized reads were then evaluated for single base-pair differences across the population.
we removed all dominant markers from the dataset because of our inability to distinguish null alleles from missing data.
using these highly conservative criteria, we identified an initial set of  <dig>  co-dominant snps with robust allele calls across the population.
linkage mapping of gpmx population
the  <dig>  codominant snps were analysed using joinmap.
they were first checked for identical pairs based on segregation data across the population and on this basis  <dig> were excluded, always dropping the snp marker with the lower quality score in each identical pair.
a further  <dig> snps were excluded as they had more than 20% missing values.
the remaining  <dig>  high confidence snps were clustered into seven linkage groups with the number of markers per group ranging from  <dig> to  <dig>  with nine remaining isolated at a lod of six.
these linkage groups were ordered using joinmap’s maximum likelihood mapping algorithm.
snps with a poor fit to the neighbouring snps were excluded and the linkage analysis was rerun leaving a total of  <dig>  unique high quality snps incorporated into the map.
the numbers of co-dominant gbs snps are presented in table  <dig>
location of six rowed spike 1
a major developmental gene, six rowed spike  <dig> , segregates in the gpmx population.
the vrs <dig> gene has previously been identified  <cit>  and profoundly affects barley spike morphology, but its effect on other plant traits such as height in gpmx is not known.
barley plants carrying a recessive vrs <dig> allele  develop spikes containing six rows of grain in contrast to the ancestral wild type spike which develops only two rows .
alternative alleles at vrs <dig> also influence the number of tillers that develop on a plant and could, as suggested previously, affect plant height, and this would influence our subsequent analysis of ari-e.gp.
the most significant associations between row type and snps were with mr_2568613p909r <dig> and mr_57812p2860r <dig>  both mapped to  <dig>  cm on chromosome 2h .
all  <dig> six-rowed lines had the same genotype as morex, while the  <dig> two-rowed lines had the same genotype as golden promise .
a major plant height qtl overlaps with the breviaristatum-e  locus
we mapped plant height as a quantitative trait for each year separately using the gpmx gbs linkage map.
a permutation test with  <dig>  permutations had a 95th percentile of  <dig>  for each year’s height data, and this was used as a genome-wide lod threshold.
this resulted in the identification of three significant plant height qtls on chromosomes 2h, 3h and 5h .
the major plant height qtl was located on chromosome 5h .
for each year’s height data, the snp most closely associated with height was mr_47526p1793r <dig> at  <dig>  cm on chromosome 5h.
this snp explained  <dig> % of the variance in height in  <dig>  with the ‘bb’ genotype having a mean height  <dig>   cm higher than the ‘aa’ genotype, and  <dig> % of the variance in height in  <dig>  with the ‘bb’ genotype having a mean height  <dig>   cm higher than the ‘aa’ genotype.
previously, it was shown that golden promise carries a mutation in the dwarfing gene known as breviaristratum-e   <cit> .
the position of the gene has been roughly estimated as about 30 cm from the short rachilla hair  locus  <cit> .
two induced mutant alleles of ari-e  and ari-e. <dig> ), were introgressed as bc6f <dig> lines into the background of cv.
‘bowman’ resulting in lines bw <dig> and bw <dig>  <cit> .
cross-referencing the snp markers that define the introgressed region in bw <dig> , which has a genetically well-defined introgression, with the barley genome sequence assembly  <cit>  supports ari-e.gp as the gene underlying the plant height qtl identified using the gpmx population.
this also supports the early observation of ari-e being linked to srh <cit> , as bw <dig>  a nearly isogenic line of cv.
bowman carrying srh, contains an introgressed segment located 10–30 cm distal to the gpmx height qtl  <cit> .
restricted multiple qtl mapping  detected two further qtls for height, the most significant markers being mr_1631678p782f <dig> at  <dig>  cm on chromosome 3h   and a region near vrs <dig>  on 2h .
for the latter, in  <dig> the most significant marker was mr_1435185p85f <dig> at  <dig>  cm while in  <dig> the most significant marker was mr_48841p1435f <dig> at  <dig>  cm.
regression analysis  was used to model the joint effects of these three locations on height.
there were no significant interactions among the three qtls, and so an additive regression on snp mr_47526p1793r <dig> from 5h, snp mr_1631678p782f <dig> from 3h and vrs1/vrs <dig> on 2h  was used.
in  <dig>  these three locations jointly explained  <dig> % of the variance in height.
for mr_1631678p782f <dig> on 3h, the ‘bb’ morex allele has a mean height  <dig>   cm higher than the ‘aa’ gp allele, and the vrs <dig> types  on 2h had a mean height  <dig>   cm lower than the vrs <dig>  types.
in  <dig>  these three locations jointly explained  <dig> % of the variance in height.
for mr_1631678p782f <dig> , the ‘bb’ allele has a mean height  <dig>   cm higher than the ‘aa’ allele and the vrs <dig> types   have a mean height  <dig>   cm lower than the vrs <dig>  types.
the effect of excluding the two lines with discrepant heights in the two years was investigated, but the qtl locations were unchanged and the differences in the parameter estimates were negligible.
what is the resolution of the gpmx gbs map at ari-e.gp?
to explore the potential of using gbs and the gpmx ril population as a platform for gene identification, we used the barley genome assembly to determine the gene content surrounding vrs <dig>  ari-e and the flanking gbs markers.
as the vrs <dig> gene is known  <cit>  we investigated the interval containing the two gbs markers that co-segregated with vrs <dig> and that fell between markers, mr_51408p1352r <dig> and mr_40453p5185r <dig>  these defined a  <dig>  cm interval on the gpmx gbs map.
current information  <cit>  indicates this interval corresponds to  <dig>  mb on the barley physical map .
it contains an estimated  <dig> genes, resulting in a gene density estimate of  <dig>  genes/mb and defining this locus as gene rich .
unlike vrs <dig>  the identity of ari-e is not known.
the qtl peak for plant height in gpmx  is associated with gbs marker mr_47526p1793r <dig> which is flanked by mr_335403p1239r <dig> and mr_1560792p1192f <dig>  these three markers define a  <dig>  cm interval on the gpmx gbs map.
however on the barley physical map marker mr_137133p6361r <dig>  appears to be positioned erroneously  making it difficult to estimate the size of the relevant interval .
replacing mr_137133p6361r <dig> with a distal marker, mr_137133p6361r <dig>  the interval defined is 46 mb and contains an estimated  <dig> genes.
we then investigated a more recent ordering of sequence contigs on the barley genome provided by the popseq methodology  <cit> .
there, the same region corresponds to a  <dig>  cm genetic interval in the barke × morex population and a  <dig>  cm interval in the oregon wolfe populations respectively.
this region contains over  <dig>  anchored sequence contigs spanning a total sequence length of approximately 10 mb  <cit> .
as the contig sequences represent only a small portion of the physical sequence, ari-e appears to be located in a relatively low recombining region.
despite residual recombination within this genetic interval, the lack of detected polymorphism suggests that the original parental haplotypes may be similar in this region and increased resolution will likely need to be sought in different populations.
CONCLUSIONS
we have shown that gbs is an effective approach for the generation of marker-dense genetic maps in cultivated barley.
the short sequence tags enabled us to directly anchor the regions containing both vrs <dig> and ari-e.gp to the recently released integrated genetic and physical sequence assembly of the barley genome and to crudely define the physical size of the two genetic intervals that we investigated.
our hope was that the flanking markers would ultimately assist us in identifying ari-e.gp.
given the resolution we obtained around ari-e.gp this seems unlikely.
our data also indicate that a region encompassing the major morphological gene vrs <dig>  which determines row-type and number of tillers in barley on 2h and an unknown locus on 3h, also affect plant height in the gpmx population.
an important practical outcome of this work for us was that we found the gbs data more challenging to handle and subsequently to analyse than the current multiplex snp assay technology we routinely run in the lab  <cit> .
indeed, this may discourage some groups from adopting the gbs approach.
nevertheless, as the principal determinant of resolution in genetic studies is a combination of the number of recombinants in the population and the number of genetic markers assayed, we were somewhat surprised  that with approximately  <dig>  informative genetic markers covering a map length of  <dig>  cm, the most closely linked markers to ari-e.gp spanned a region of approximately 7 cm.
it is possible that the mutant ari-e.gp locus was induced within a local haplotype that is shared with morex .
indeed, evidence from the barley 9 k iselect snp genotyping platform on the parents indicates that gp and morex probably do share a common haplotype across the proximal short arm and centromeric region of 5h.
this would unfortunately result in a markerless gap in the genetic map.
similar gaps have been evident in other high-density genetic maps of barley  <cit> .
despite this, the flanking snp-containing gbs tags will be easy to convert to single-locus markers, and these will be highly valuable for identifying additional recombinants around ari-e.gp and, if pursued further using a map based approach, ultimately the identification of the gene.

 barley is a diploid economically important cereal crop and genetic model for small grain temperate cereals. golden promise is a two-rowed uk spring barley cultivar, and is currently the most responsive genotype for barley genetic transformation. also, because of its unique properties, the malt extracted from gp is used to distil a number of signature single malt scotch whiskies such as macallan and glengoyne. it is a primary induced gamma-ray mutant derivative of the barley cultivar maythorpe, and is known to contain a mutation in breviaristatum-e. this mutation in ari-e in gp causes a semi-dwarfing phenotype that has been used widely in barley cultivar development to shorten straw length and reduce the severity of lodging. gp is also susceptible to several fungal pathogens, has short awns, reduced internode length and shows a measure of tolerance to salt. genetic analysis has previously located ari-e.gp to barley chromosome 5h as a quantitative trait locus influencing plant height, and physiological studies have confirmed its relative insensitivity to the addition of exogenous gibberellic acid. the ari-e gene has not yet been cloned although it was recently mapped as a height qtl using the tools of contemporary biometrical genetics in a complex three-way cross. over the past two decades, many molecular tools have been developed in barley to enable genetic research. the primary focus has been the construction of molecular marker-based genetic linkage maps that can be leveraged for mapping genes of interest and subsequent marker assisted selection in breeding programs. these have been applied to discover, dissect and manipulate genes determining a range of simple and complex traits. because of their value, accompanied by their increasing use in genetics and breeding, there has been a continual drive to both reduce marker costs and to avoid ascertainment issues while at the same time enhancing flexibility and marker throughput per assay. it is therefore appropriate that new developments in marker technology are both explored and thoroughly evaluated against the current state of the art. now that next generation sequencing technology has been shown to be capable of discovering and genotyping thousands of markers across almost any genome of interest at low cost and in a single step, a current debate is whether sequence-based genotyping methods are ready to replace many of the established and widely used tools such as highly-multiplex single nucleotide polymorphism platforms. available sequence-based genotyping methods generally rely upon the use of restriction enzymes to produce a reduced representation of the non-repetitive regions of the genome. restriction site-associated genomic dna typing is such an approach and has been used in several species for the construction of linkage maps and application in qtl analyses. in barley, a rad linkage map was recently produced in a double haploid population and used for qtl analysis. elshire and colleagues subsequently described a similar but more straightforward method of genotyping by sequencing which works effectively in 96-well plate assays. gbs was originally developed for high-resolution association studies in maize and, like rad, has been extended to a range of species with complex genomes. a two-enzyme gbs protocol has now been developed that produces a uniform library for sequencing and has been applied to both wheat and barley. this gbs approach has been shown to be suited to genetic analysis of rapeseed, lupin, lettuce, switchgrass, soybean, and maize. in this report, our biological objective was to identify at high resolution the genetic location of the ari-e.gp semi-dwarfing gene of cultivated barley. however, as a sequence assembly of the barley genome has just been published, we also wanted to use a sequence-based genetic marker methodology that would in principle allow us to link directly to the genome sequence assemblies and physical map, ultimately as a shortcut to facilitate the identification of the ari-e gene. we therefore chose to explore use of the two-enzyme based gbs method, using digestion of genomic dna with a six-base methylation sensitive ‘rare-cutter’ and a four-base ‘common cutter’ enzyme. we combined this with the illumina ngs platform and developed a downstream informatics pipeline to discover co-dominant in an f single-seed descent mapping population from a golden promise by morex cross. in contrast to gp, mx is a tall spring six-rowed north american barley variety with desirable malting and brewing characteristics. most importantly, mx is the reference cultivar used in the barley genome sequencing efforts. our genetic analysis using gbs data from the recombinant inbred population confirmed the location of ari-e.gp on barley chromosome 5h. in the process we discovered high-confidence snps that we could associate with contigs in the ngs sequence assemblies and physical map.  golden promise by morex population and variation in plant height a recombinant inbred line population of 160 f single-seed descent lines from a gp by mx cross was developed over seven years, from 2003– at the james hutton institute. the 136 f rils used in this study comprised two-rowed and six-rowed accessions. the population segregates quantitatively for height as shown in figure  varying between different rils from to 130 cm. asymmetric transgressive segregation of plant height across the gpmx population can be observed – there were around lines taller than morex, but only about shorter than golden promise. the pearson correlation between the heights in the two years was. two lines showed a marked difference in height between the two years. generation of psti reference sequences from barley genome assemblies to facilitate genetic analysis by gbs we first extracted a set of 64 bp reference sequences flanking all predicted psti restriction sites from barley genome assemblies of the cultivars morex ), bowman and barke using the ‘restrict’ program from the emboss suite of tools. for cultivar morex, restriction sites were identified, yielding a total of gbs reference sequences present on unique morex genome assembly contigs. of all the identified sites, flanking sequences had to be excluded because the restriction site was too close to the start or end of an assembled genomic contig and therefore extraction of the full 64 bp sequence was impossible. extraction of additional sequences unique to the genome assemblies of cultivars bowman and barke yielded a further and sequences, respectively. after removal of chloroplast sequences a total of reference sequences remained that were subsequently used for read mapping. more than half of the 64 bp reference sequences stemmed from morex contigs that contain regions of homology to full length cdnas or expressed genes, previously mapped genetic markers or sequences that have chromosome arm assignments based on survey sequencing of flow sorted chromosome arms. gbs reads of gpmx we generated three 48-plex gpmx gbs libraries representing all progenies and the parents, which were repeatedly represented in each library for qc purposes. we used psti combined with msei to digest genomic dna, with the psti overhang sequence located in the barcode adapter adjacent to the barcode sequence, and the msei overhang sequence located in common y-adapter. single-end sequencing starting from the barcoded adapter was performed using illumina chemistry. pilot sequencing the gpmx_ library on an illumina gaii platform generated more than million single-end reads of 72 bp in length. of these, over 58 m reads were categorised as having a correct barcode and psti overhang sequence. further sequencing of all three gpmx population libraries, each on one lane of an illumina hiseq generated a total of 622 m reads of 100 bp and more than 482 m remaining as categorised reads. the average number of categorised reads obtained per lane was  m on the illumina ga ii and 160 m on the illumina hiseq by applying various filtering criteria in the reads), the percentages of categorised reads were % and %. after deconvolution the distribution of the number of reads per sample ranged from to,. the read distribution was relatively even across the population, with only of the lines having less than a million reads. for the parents, we obtained  m reads from golden promise and  m from morex, due to repeat sequencing. all sequence reads generated from gpmx were submitted to the sequence read archive section of the european nucleotide archive. co-dominant markers from the gpmx population datasets in total, 461 m categorized reads from the gpmx mapping population were mapped to the 64 bp reference sequences using the bowtie mapping tool. in order to reduce the number of false positive snps during downstream analysis, only a single mismatch per read was allowed, and only uniquely mapped reads were included, leaving 46% of 461 m reads mapped to the reference. these categorized reads were then evaluated for single base-pair differences across the population. we removed all dominant markers from the dataset because of our inability to distinguish null alleles from missing data. using these highly conservative criteria, we identified an initial set of co-dominant snps with robust allele calls across the population. linkage mapping of gpmx population the codominant snps were analysed using joinmap. they were first checked for identical pairs based on segregation data across the population and on this basis were excluded, always dropping the snp marker with the lower quality score in each identical pair. a further snps were excluded as they had more than 20% missing values. the remaining high confidence snps were clustered into seven linkage groups with the number of markers per group ranging from to with nine remaining isolated at a lod of six. these linkage groups were ordered using joinmap’s maximum likelihood mapping algorithm. snps with a poor fit to the neighbouring snps were excluded and the linkage analysis was rerun leaving a total of unique high quality snps incorporated into the map. the numbers of co-dominant gbs snps are presented in table  location of six rowed spike 1 a major developmental gene, six rowed spike, segregates in the gpmx population. the vrs gene has previously been identified and profoundly affects barley spike morphology, but its effect on other plant traits such as height in gpmx is not known. barley plants carrying a recessive vrs allele develop spikes containing six rows of grain in contrast to the ancestral wild type spike which develops only two rows. alternative alleles at vrs also influence the number of tillers that develop on a plant and could, as suggested previously, affect plant height, and this would influence our subsequent analysis of ari-e.gp. the most significant associations between row type and snps were with mr_2568613p909r and mr_57812p2860r both mapped to  cm on chromosome 2h. all six-rowed lines had the same genotype as morex, while the two-rowed lines had the same genotype as golden promise. a major plant height qtl overlaps with the breviaristatum-e locus we mapped plant height as a quantitative trait for each year separately using the gpmx gbs linkage map. a permutation test with permutations had a 95th percentile of for each year’s height data, and this was used as a genome-wide lod threshold. this resulted in the identification of three significant plant height qtls on chromosomes 2h, 3h and 5h. the major plant height qtl was located on chromosome 5h. for each year’s height data, the snp most closely associated with height was mr_47526p1793r at  cm on chromosome 5h. this snp explained % of the variance in height in with the ‘bb’ genotype having a mean height cm higher than the ‘aa’ genotype, and % of the variance in height in with the ‘bb’ genotype having a mean height cm higher than the ‘aa’ genotype. previously, it was shown that golden promise carries a mutation in the dwarfing gene known as breviaristratum-e. the position of the gene has been roughly estimated as about 30 cm from the short rachilla hair locus. two induced mutant alleles of ari-e and ari-e. ), were introgressed as bc6f lines into the background of cv. ‘bowman’ resulting in lines bw and bw. cross-referencing the snp markers that define the introgressed region in bw, which has a genetically well-defined introgression, with the barley genome sequence assembly supports ari-e.gp as the gene underlying the plant height qtl identified using the gpmx population. this also supports the early observation of ari-e being linked to srh, as bw a nearly isogenic line of cv. bowman carrying srh, contains an introgressed segment located 10–30 cm distal to the gpmx height qtl. restricted multiple qtl mapping detected two further qtls for height, the most significant markers being mr_1631678p782f at  cm on chromosome 3h and a region near vrs on 2h. for the latter, in the most significant marker was mr_1435185p85f at  cm while in the most significant marker was mr_48841p1435f at  cm. regression analysis was used to model the joint effects of these three locations on height. there were no significant interactions among the three qtls, and so an additive regression on snp mr_47526p1793r from 5h, snp mr_1631678p782f from 3h and vrs1/vrs on 2h was used. in these three locations jointly explained % of the variance in height. for mr_1631678p782f on 3h, the ‘bb’ morex allele has a mean height cm higher than the ‘aa’ gp allele, and the vrs types on 2h had a mean height cm lower than the vrs types. in these three locations jointly explained % of the variance in height. for mr_1631678p782f, the ‘bb’ allele has a mean height cm higher than the ‘aa’ allele and the vrs types have a mean height cm lower than the vrs types. the effect of excluding the two lines with discrepant heights in the two years was investigated, but the qtl locations were unchanged and the differences in the parameter estimates were negligible. what is the resolution of the gpmx gbs map at ari-e.gp? to explore the potential of using gbs and the gpmx ril population as a platform for gene identification, we used the barley genome assembly to determine the gene content surrounding vrs ari-e and the flanking gbs markers. as the vrs gene is known we investigated the interval containing the two gbs markers that co-segregated with vrs and that fell between markers, mr_51408p1352r and mr_40453p5185r these defined a  cm interval on the gpmx gbs map. current information indicates this interval corresponds to  mb on the barley physical map. it contains an estimated genes, resulting in a gene density estimate of genes/mb and defining this locus as gene rich. unlike vrs the identity of ari-e is not known. the qtl peak for plant height in gpmx is associated with gbs marker mr_47526p1793r which is flanked by mr_335403p1239r and mr_1560792p1192f these three markers define a  cm interval on the gpmx gbs map. however on the barley physical map marker mr_137133p6361r appears to be positioned erroneously making it difficult to estimate the size of the relevant interval. replacing mr_137133p6361r with a distal marker, mr_137133p6361r the interval defined is 46 mb and contains an estimated genes. we then investigated a more recent ordering of sequence contigs on the barley genome provided by the popseq methodology. there, the same region corresponds to a  cm genetic interval in the barke × morex population and a  cm interval in the oregon wolfe populations respectively. this region contains over anchored sequence contigs spanning a total sequence length of approximately 10 mb. as the contig sequences represent only a small portion of the physical sequence, ari-e appears to be located in a relatively low recombining region. despite residual recombination within this genetic interval, the lack of detected polymorphism suggests that the original parental haplotypes may be similar in this region and increased resolution will likely need to be sought in different populations.  we have shown that gbs is an effective approach for the generation of marker-dense genetic maps in cultivated barley. the short sequence tags enabled us to directly anchor the regions containing both vrs and ari-e.gp to the recently released integrated genetic and physical sequence assembly of the barley genome and to crudely define the physical size of the two genetic intervals that we investigated. our hope was that the flanking markers would ultimately assist us in identifying ari-e.gp. given the resolution we obtained around ari-e.gp this seems unlikely. our data also indicate that a region encompassing the major morphological gene vrs which determines row-type and number of tillers in barley on 2h and an unknown locus on 3h, also affect plant height in the gpmx population. an important practical outcome of this work for us was that we found the gbs data more challenging to handle and subsequently to analyse than the current multiplex snp assay technology we routinely run in the lab. indeed, this may discourage some groups from adopting the gbs approach. nevertheless, as the principal determinant of resolution in genetic studies is a combination of the number of recombinants in the population and the number of genetic markers assayed, we were somewhat surprised that with approximately informative genetic markers covering a map length of  cm, the most closely linked markers to ari-e.gp spanned a region of approximately 7 cm. it is possible that the mutant ari-e.gp locus was induced within a local haplotype that is shared with morex. indeed, evidence from the barley 9 k iselect snp genotyping platform on the parents indicates that gp and morex probably do share a common haplotype across the proximal short arm and centromeric region of 5h. this would unfortunately result in a markerless gap in the genetic map. similar gaps have been evident in other high-density genetic maps of barley. despite this, the flanking snp-containing gbs tags will be easy to convert to single-locus markers, and these will be highly valuable for identifying additional recombinants around ari-e.gp and, if pursued further using a map based approach, ultimately the identification of the gene. 
using gbs mapping data and phenotypic measurements we show that ari-e.gp maps to a small genetic interval on chromosome 5h and that alternative alleles at a region encompassing vrs on 2h along with a region on chromosome 3h also influence plant height. barley cultivars in north-western europe largely contain either of two dwarfing genes; denso on chromosome 3h, a presumed ortholog of the rice green revolution gene ossd or breviaristatum-e on chromosome 5h. gbs was an effective and relatively low-cost approach to rapidly construct a genetic map of the gpmx population that was suitable for genetic analysis of row type and height traits, allowing us to precisely position ari-e.gp on chromosome 5h. we found the gbs data more complex to analyse than other data types but it did directly provide linked snp markers for subsequent higher resolution genetic analysis. we explored use of the gbs to populate the region with sequence contigs from the recently released physically and genetically integrated barley genome sequence assembly as a step towards ari-e gene identification.
 barley cultivars in north-western europe largely contain either of two dwarfing genes; denso on chromosome 3h, a presumed ortholog of the rice green revolution gene ossd or breviaristatum-e on chromosome 5h. a recessive mutant allele of the latter gene, ari-e.gp, was introduced into cultivation via the cv. ‘golden promise’ that was a favourite of the scottish malt whisky industry for many years and is still used in agriculture today.  using gbs mapping data and phenotypic measurements we show that ari-e.gp maps to a small genetic interval on chromosome 5h and that alternative alleles at a region encompassing vrs on 2h along with a region on chromosome 3h also influence plant height. the location of ari-e is supported by analysis of near-isogenic lines containing different ari-e alleles. we explored use of the gbs to populate the region with sequence contigs from the recently released physically and genetically integrated barley genome sequence assembly as a step towards ari-e gene identification.  gbs was an effective and relatively low-cost approach to rapidly construct a genetic map of the gpmx population that was suitable for genetic analysis of row type and height traits, allowing us to precisely position ari-e.gp on chromosome 5h. mapping resolution was lower than we anticipated. we found the gbs data more complex to analyse than other data types but it did directly provide linked snp markers for subsequent higher resolution genetic analysis. barleydwarfing genegenotyping by sequencingphysical map 