BACKGROUND
preharvest sprouting is observed across all major wheat growing regions in the world.
in western canada, the average annual losses due to phs are approximately $ <dig> million  <cit> .
insufficient seed dormancy is one major factor contributing to pre-harvest sprouting losses, particularly under humid, wet weather conditions at harvest.
phs resistant/tolerant wheat cultivars and land races have been identified globally, with origins mainly in canada, usa, australia, china, japan, south africa, kenya and new zealand  <cit> .
canadian red-seeded spring wheat cultivars  and white spring wheat genotypes  are known to carry resistance to phs, all having derived their resistance alleles from a red-seeded breeding line rl <dig>  <cit> .
of the three phs traits, fn  <cit>  is most commonly used to quantify phs  <cit>  and indirectly measures the activity of the enzyme α-amylase that breaks down starch in germinating grains.
degradation of grain-starch as the result of greater α-amylase activitys result in lower fn values and are an indirect indication of low levels of phs resistance or dormancy.
two other important traits for the characterization of phs are gi  <cit>  and si  <cit> .
while gi values deduced from seed-germination tests in petri dishes are a direct measure of seed dormancy, si values obtained via artificial wetting of intact wheat spikes, detect dormancy and properties of the inflorescence that affect phs  <cit> .
quantitative trait loci  linked to phs traits have been reported on all  <dig> hexaploid wheat chromosomes  <cit> , mainly on 3a  <cit> , 3b and 3d  <cit> , 4a  <cit> , 5a  <cit> , 6b and 7d  <cit> .
of these, the phs qtl on 4a has consistently been identified in several different mapping populations.
the rl4452/‘ac domain’ dh population has been extensively characterized for qtl detection of phs  <cit> , agronomic  <cit>  and quality traits  <cit> , in several past studies that involved a small number of molecular markers.
these studies relied mainly on ssr marker data for the preparation of genetic maps and locating qtl on chromosomes.
with low costs and rapid advancements in sequencing technology, thousands of molecular markers, mainly snps have become available in wheat.
additionally, access to genome sequence information for rice  <cit>  and brachypodium  <cit>  will now facilitate comparative mapping for the identification of genes underlying various important quantitative traits in wheat.
interaction among phs qtl , and the environment  have been reported from various studies  <cit>  aimed at understanding the complex genetic structure of qtl.
as chromosomal locations of phs qtl are not uniform across populations, obtaining a consensus on the precise genomic location of important trait qtl is required for fine mapping and cloning studies.
meta-qtl or meta-analysis  <cit>  integrates several qtl studies of a common trait to provide a meaningful estimate of the exact location and number of qtl for that given trait.
eight phs qtl on chromosomes 3a, 3b, 3d and 4a were identified in a meta-qtl study  <cit>  involving  <dig> different populations .
a high level of genome-synteny exists among wheat, brachypodium and rice, with wheat being more closely related to brachypodium than to rice  <cit> .
conservation or collinearity of genetic markers  <cit>  and greater structural similarities in the coding regions of orthologous genes  <cit>  of wheat and brachypodium have been reported.
however, given differences in gene content in orthologous regions of wheat, brachypodium  <cit>  and rice  <cit> , it might be beneficial to use both genomic sequences of brachypodium and rice in comparative mapping studies for map based cloning and gene discovery in wheat.
our study deployed snp markers from a 90k infinium iselect custom beadchip  <cit> , in addition to available ssr, dart and ests, to generate high density genetic maps for the identification of phs resistance qtl.
sequences corresponding to polymorphic snps flanking phs qtl were analyzed against genomic sequences of brachypodium and rice.
the objectives of our research were a) to identify candidate genes and regions in brachypodium and rice that are orthologous to phs resistance qtl intervals in wheat, and b) to utilize sequences of snps flanking phs qtl to develop kasp markers for mas of phs resistance.
RESULTS
linkage mapping
a total of  <dig>  snp, ssr, dart and est markers were mapped to all  <dig> wheat chromosomes.
the resulting linkage map spanning  <dig>  cm is reported in additional file  <dig>  of the  <dig>  markers,  <dig>  or  <dig> % were snps, while the remaining  <dig> or  <dig> % comprised ssr, dart and est markers.
the largest number of snp markers  were distributed across the b genome, followed by  <dig>  snps mapped to the a genome, and  <dig>  snp markers on the d genome .table 1table  <dig>
cumulative map-lengths of a, b and d genome chromosomes alongside corresponding genome-wise distribution of snp markers mapped in the hexaploid dh population of rl4452/‘ac domain’
genome
map length
mapped markers
snps
ssrs, darts & ests
% snps
qtl analysis
phs datasets were analyzed with both mim and simple interval mapping  methods.
as results of both methods were very similar, only those of mim were reported in this study.
the mim  <cit>  analysis identified four qtl with significant effects, located across chromosomes 3b, 4a, 7b and 7d.
each of these four qtl appeared in two or more environments and had peak lod scores greater than the critical threshold lod at 5% significance levels   <cit> .
coincident qtl for gi, si and fn were located on chromosome 4a.
across trials, rl <dig> alleles on 3b and 7b provided phs resistance as they reduced si.
however, ‘ac domain’ alleles also provided phs resistance as they increased fn on 4a and 7d  and reduced si and gi on 4a .table  <dig>
results of multiple interval mapping : four qtl for phs traits  identified on chromosomes 3b, 4a, 7b.
<dig> and 7d.
<dig> in a dh population of rl4452/‘ac domain’ replicated in multi-year environments
qtl
trial dataset
chromosome
qtl peak location
additive
a
% pv
lod
α <dig>
germination index
qgi.crc-4a
glenlea2005
qgi.crc-4a
winnipeg2004
qgi.crc-4a
winnipeg2005
sprouting index
qsi.crc-3b
glenlea2005
qsi.crc-3b
winnipeg2004
qsi.crc-4a
glenlea2005
qsi.crc-4a
winnipeg2004
qsi.crc-4a
winnipeg2005
qsi.crc-4a
swift current2003
qsi.crc-7b
swift current2003
qsi.crc-7b
swift current2004
falling number
qfn.crc-4a
glenlea2005
qfn.crc-4a
winnipeg2004
qfn.crc-7d
glenlea2003
qfn.crc-7d
glenlea2005
apositive or negative additive values relate to allele effects of the ac domain parent.
candidate regions and genes for phs resistance
sequences of snps flanking qtl for phs resistance on chromosomes 3b, 4a, 7b and 7d were subjected to blastn searches on the iwgsc and gramene databases and returned hits to candidate regions in brachypodium and rice .
genetic and physical maps displaying orthologous regions for phs resistance in wheat, brachypodium and rice are given in figures 1a and b. a  <dig>  cm qtl interval on chromosome 3b was orthologous to a ~ <dig>  mb region  on chromosome  <dig> of brachypodium  and to a ~ <dig>  mb  region on chromosome  <dig> of rice .
the 4a qtl interval was  <dig>  cm and was orthologous to a ~ <dig>  mb region  on chromosome  <dig> of brachypodium  and to a ~ <dig>  mb  region on chromosome  <dig> of rice .
on chromosome 7b.
<dig>  the qtl interval spanned  <dig>  cm and was orthologous to a ~ <dig>  mb region  on chromosome  <dig> of brachypodium  and to a ~1 mb  region on chromosome  <dig> of rice .
the qtl interval on 7d.
<dig> was  <dig>  cm and was orthologous to a ~ <dig>  mb region  on chromosome  <dig> of brachypodium , and a ~ <dig>  mb region  on chromosome  <dig> of rice .table  <dig>
genetic map locations of snp markers flanking phs qtl on chromosomes 3b, 4a, 7b.
<dig> and 7d.
<dig> in a wheat dh population of a rl4452/‘ac domain’ cross and their corresponding physical locations/candidate regions in
brachypodium distachyon
and rice
snp marker
map
survey sequence
blastn hits to
brachypodium
genes
blastn hits to rice genes
contig no.
chromosome 3b
chromosome 4a
chromosome 7b.1
chromosome 7d.2
!weak hit to genomic regions in brachpodium or rice that is orthologous to the qtl interval for phs resistance in wheat.
best hits that do not correspond to the candidate region in brachpodium or rice are in italics.
location of qtl and syntenic regions in
brachpodium
and rice.
a. location of qtl and flanking markers for phs resistance on a) wheat chromosome 3b and its candidate regions on brachypodium bd <dig> and rice os <dig>  and b) chromosome 7b.
<dig> and its candidate regions on brachypodium bd <dig> and rice os <dig>
b. location of qtl and flanking markers for phs resistance on a) wheat chromosome 4a and its candidate regions on brachypodium bd <dig> and rice os <dig>  and b) chromosome 7d.
<dig> and its candidate regions on brachypodium bd <dig> and rice os <dig>
brachypodium/rice candidates for phs resistance orthologous to consensus regions on wheat chromosomes 3b, 4a, 7b and 7d  were identified.
in the 3b region there are  <dig> genes in the brachypodium orthologous region and  <dig> in the rice region.
the 4a region had  <dig> genes in the brachypodium region and  <dig> in rice, while the 7b region had  <dig> in brachypodium and  <dig> in rice and the 7d region had  <dig> in brachypodium and  <dig> in rice.
genes involved in aba and ga metabolism as well as those affecting flowering time were present in the qtl regions.
among these were bradi2g49795/os01g <dig> , os01g <dig>  os01g <dig>  orthologous to chromosome 3b, bradi1g00950/os03g <dig> , os03g <dig>  os03g <dig>  orthologous to 4a, bradi1g46060/os06g <dig>  orthologous to 7b, bradi1g <dig>  bradi1g <dig>  bradi1g <dig>  bradi1g <dig> , bradi1g <dig>  bradi1g <dig>  and bradi1g48830/os06g <dig>  orthologous to chromosome 7d.
development and validation of kasp primers
a total of  <dig> kasp markers, five each for chromosomes 3b and 7b.
<dig>  and four each for chromosomes 4a and 7d.
<dig>  were developed from sequences of snps flanking qtl for phs resistance.
genetic map locations of individual kasp markers were identical to the respective snp from which they were derived.
primer sets of all  <dig> kasp markers are listed in additional file  <dig>  further, we validated the conversion of these  <dig> kasps from matching genotype calls of infinium snp data on  <dig> dh progeny genotypes.
four dh progeny genotypes of the rl4452/‘ac domain’ cross were identified to carry phs resistance on chromosomes 3b, 4a, 7b and 7d .table  <dig>
a list of  <dig> competitive allele-specific pcr  markers developed for mas of phs from snps flanking phs qtl on chromosomes 3b, 4a, 7b and 7d in a dh population of a rl4452/‘ac domain’ cross
sl.
kasp marker
source snp
chr
phs trait
si
si
si
si
si
gi, si, fn
gi, si, fn
gi, si, fn
gi, si, fn
si
si
si
si
si
fn
fn
fn
fn
discussion
the objectives of our research were to identify candidate regions for phs resistance qtl of wheat and develop kasp markers  from sequences of snps flanking such qtl.
this is an important step in the process of map-based cloning of genes that underlie important quantitative traits like phs resistance.
our objectives were achieved using  <dig>  snps from the 90 k infinium custom beadchip to develop a high density linkage map in the rl4452/‘ac domain’ mapping population and subsequently detect qtl for phs resistance on chromosomes 3b, 4a, 7b and 7d.
comparative mapping utilizing sequences of snps flanking phs resistance qtl enabled identification of candidate genes and regions in brachypodium and rice.
the resulting  <dig> kasp markers can be deployed in future genetic studies of phs, and in evaluation of phs in germplasm and breeding material.
of the  <dig>  mapped markers,  <dig>  or  <dig> % were snp markers, while the remaining  <dig> or  <dig> % were ssr, dart and est markers.
the b genome chromosomes accounted for the largest number of  <dig> snp markers, followed by the a genome with  <dig> snps, and the d genome with  <dig> snp markers.
a likely explanation for larger numbers of b genome snp markers could be the greater genetic diversity of b genome species when compared to the a and d genome species  <cit> .
a faster rate of evolution of the b genome due to greater polymorphism and duplication events, in addition to greater genetic diversity brought about by cross pollination were cited  <cit>  as possible explanations for findings of a greater number of ests associated with more unique loci on the b genome when compared to the a and d genomes.
phs datasets were analyzed with both mim and sim  methods.
because results of both methods were very similar, only those of the mim analyses were reported.
as qtl identified using mim were robust and supported by sim results, it is unlikely that additional large effect qtl involved in epistatic interactions might have been detected using other qtl mapping methods that detect both main effect  and epistatic qtl .
further, a meta-qtl study  <cit>  reporting phs qtl on 4a and group  <dig> chromosomes support significant phs qtl identified on chromosome 3b and 4a of our study.
the most consistent of the four phs qtl identified on chromosomes 3b, 4a, 7b and 7d were located on chromosome 4a; gi, si and fn trait qtl each accounting for  <dig> %,  <dig> % and  <dig> % of the phenotypic variation in their respective traits.
the qtl for these phs traits were coincident and maybe associated with the same gene.
these findings might suggest that chromosome 4a is involved in regulation of phs trait qtl in our test population.
previous reports of the association of phs traits with chromosome 4a  <cit> , support the importance of this qtl for phs
in addition to a major si qtl on 4a, two other qtl for si were identified on chromosomes 3b and 7b.
<dig>  both si qtl on 3b and 7b.
<dig> were detected in two of six environments.
qtl that provide tolerance to late maturity α- amylase  have been mapped on 3bs and 7bl in an australian wheat cross cranbrook/halberd  <cit> .
in both studies, the ssr markers xwmc <dig>  xwmc <dig>  xgwm <dig>  xwmc <dig>  xgwm <dig>  xwmc <dig>  xwmc <dig>  and xgwm <dig>  xwmc <dig>  xwmc <dig>  also flanked corresponding phs qtl intervals on chromosomes 3b.
<dig> and 7b.
<dig> respectively .
further, alleles of a regulator gene vp-1b on 3b have been reported to influence grain dormancy in chinese wheat varieties  <cit> .
in a follow up study  <cit> , the vp-1b locus was validated in a white-grained chinese landrace wanxianbaimaizi  using ssr markers and a gene-specific primer vp <dig>  a cim analysis identified a seed dormancy qtl qsd.ahau-3b on 3b flanked by vp <dig> which is linked to an ssr marker xwmc <dig> that also happens to flank the phs qtl interval on chromosome 3b of our study.
the above findings suggest that phs and lma qtl on chromosomes 3b and 7b are likely the same.
‘ac domain’ alleles contributed to increasing the fn on 7d , with the exception of the glenlea  <dig> trial, wherein a negative additive score was observed for the fn.
while the fn qtl on chromosome 7d is unique to our study, a significant time to maturity  qtl  also on 7d, and a positive contribution of the rl <dig> allele, has been reported previously by  <cit>  in the same rl4452/‘ac domain’ population.
the authors reported an ssr marker xgwm <dig> tightly linked to this qtl, which is distally located on 7dl, and is  <dig>  cm from the qtl peak of our study.
in the glenlea  <dig> trial , the average fn  score of  <dig> dh progeny was the lowest of the four trials .
the low fn score at this location might suggest greater levels of phs of ‘ac domain’ genotypes, probably brought on by wet weather conditions at the maturity stages or during the three weeks preceding harvest  <cit> .
as qtl locations of both these mat and fn traits nearly coincide and are influenced by negative and positive additive effects  of ‘ac domain’ alleles respectively, the action of a pleiotrophic locus regulating both fn and mat could be assumed.
at glenlea in  <dig> it is possible that the lower fn for the domain allele is due to adverse weather conditions at maturity or that the 7d qtl identified here might not actually be a phs qtl, but rather a pleiotrophic effect of the mat qtl on phs.
flanking marker intervals of a given phs trait  qtl were not always the same across trials/datasets.
it is quite likely that the respective underlying genes influencing each of these traits are the same; difference in qtl interval location being mainly due to environment or experimental error from differences in class means of individual trial data sets  <cit> .
alternatively, the possibility of two closely linked loci controlling the same trait cannot be ruled out.
blastn searches with sequences of snp markers flanking phs qtl on chromosomes 3b, 4a, 7b and 7d revealed candidate regions in brachypodium and rice genomes.
the qtl interval on chromosome 3b was orthologous to regions on bradi <dig> and the long arm of os <dig>  while qtl intervals on chromosomes 4a were orthologous to regions on bradi <dig> and the short arm of os <dig>  qtl intervals on chromosome 7b.
<dig> and 7d.
<dig> were orthologous to regions on bradi <dig> and the short arm of os <dig> of rice.
the above findings of orthology between wheat/rice chromosomes: 3b/os <dig>  4a/os <dig> and 7b&7d/os <dig> concur with previous reports  <cit>  of wheat/rice chromosomal region similarities revealed via comparative mapping with dna probes and ests.
further, orthologies between phs qtl intervals of 4a, 7b, 7d and genomic regions of bradi <dig>  and 3b/bradi <dig> in our study will be refined to tease out individual genes responsible for variation in phs resistance.
the availability of information on whole-genome  <dig> assembled gene sequences of chinese spring  <cit>  and gene-orthologies among the said wheat and brachypodium chromosomes established using  <dig> ests mapped to wheat deletion bins  <cit>  will serve as useful references to complement our efforts.
eighteen kasp markers were developed from snp sequences flanking qtl for phs resistance.
identical genotype calls of infinium snp data enabled validation of the  <dig> kasp markers and identified four  progeny genotypes of the rl4452/‘ac domain’ population possessing phs resistance on all four qtl on 3b, 4a, 7b and 7d .
criteria for selection of these genotypes was based on findings of our study: ‘ac domain’  reduced gi and si on 4a, increased fn on 4a and 7d, while rl <dig>  reduced si on chromosomes 3b and 7b.
further, these  <dig> kasp markers can be deployed in future genetic studies of phs, and in evaluation of phs in germplasm and breeding material.
genes present in brachypodium and rice in orthologous regions corresponding to the qtl were identified .
the 3b region is large and contains over  <dig> genes in brachypodium and over  <dig> in rice.
more markers are needed to reduce the size of the region and the emerging reference sequence of chromosome 3b  will be a valuable resource.
there are a number of aba-inducible genes  which could be a starting point to search for additional markers.
the 4a and 7b regions contain many fewer genes in brachypodium and rice than the 3b region.
gibberellin  <dig> oxidase   <cit>  on bradi1/os <dig> orthologous to 4a and abscissic acid responsive elements   <cit>  on bradi1/os <dig> orthologous to chromosome 7b are candidates worth further study.
ga <dig> - oxidase has previously been considered as a candidate gene underlying phs qtl on 4a  <cit> .
on chromosome 7d the qtl was coincident with a previously identified maturity qtl in the same population .
genes affecting flowering time are present in the orthologous regions in brachypodium and rice.
these include the far-red impaired responsive  related proteins  <cit>  on chromosome bradi <dig>  as well as vrn1-ap2/b3-like transcription factors  <cit>  on bradi <dig> and phosphatidylethanolamine - binding protein  homologous to the flowering locus t gene  <cit>  on bradi1/os <dig>  orthologous to chromosome 7d.
because our study utilized a large number of sequence-based snps not available for previous mapping studies, the resulting genetic maps and qtl flanking snp markers are a novel and current resource for identification of underlying genes based on synteny and collinearity to model species brachypodium and rice.
further, the identification of candidate genes and regions for phs in brachypodium and rice will enable a targeted focus for selection of candidate genes whose physiological/biological functions are linked to or influence variation in phs traits under study.
such candidate gene-specific pcr markers will be developed and validated via mapping to the qtl intervals for phs resistance in wheat.
CONCLUSIONS
in our study we utilized snps from a wheat 90 k infinium iselect custom beadchip that permitted detection and assignment of significant phs resistance qtl to specific chromosomal locations on genetic maps.
sequences of snps flanking phs resistance qtl enabled identification of candidate genes and regions for phs in brachypodium and rice via comparative mapping.
the  <dig> kasp markers resulting from this study can be suitably deployed in future genetic studies of phs and might also be useful in the evaluation of phs in germplasm and breeding material.

 preharvest sprouting is observed across all major wheat growing regions in the world. in western canada, the average annual losses due to phs are approximately $ million. insufficient seed dormancy is one major factor contributing to pre-harvest sprouting losses, particularly under humid, wet weather conditions at harvest. phs resistant/tolerant wheat cultivars and land races have been identified globally, with origins mainly in canada, usa, australia, china, japan, south africa, kenya and new zealand. canadian red-seeded spring wheat cultivars and white spring wheat genotypes are known to carry resistance to phs, all having derived their resistance alleles from a red-seeded breeding line rl. of the three phs traits, fn is most commonly used to quantify phs and indirectly measures the activity of the enzyme α-amylase that breaks down starch in germinating grains. degradation of grain-starch as the result of greater α-amylase activitys result in lower fn values and are an indirect indication of low levels of phs resistance or dormancy. two other important traits for the characterization of phs are gi and si. while gi values deduced from seed-germination tests in petri dishes are a direct measure of seed dormancy, si values obtained via artificial wetting of intact wheat spikes, detect dormancy and properties of the inflorescence that affect phs. quantitative trait loci linked to phs traits have been reported on all hexaploid wheat chromosomes, mainly on 3a, 3b and 3d, 4a, 5a, 6b and 7d. of these, the phs qtl on 4a has consistently been identified in several different mapping populations. the rl4452/‘ac domain’ dh population has been extensively characterized for qtl detection of phs, agronomic and quality traits, in several past studies that involved a small number of molecular markers. these studies relied mainly on ssr marker data for the preparation of genetic maps and locating qtl on chromosomes. with low costs and rapid advancements in sequencing technology, thousands of molecular markers, mainly snps have become available in wheat. additionally, access to genome sequence information for rice and brachypodium will now facilitate comparative mapping for the identification of genes underlying various important quantitative traits in wheat. interaction among phs qtl, and the environment have been reported from various studies aimed at understanding the complex genetic structure of qtl. as chromosomal locations of phs qtl are not uniform across populations, obtaining a consensus on the precise genomic location of important trait qtl is required for fine mapping and cloning studies. meta-qtl or meta-analysis integrates several qtl studies of a common trait to provide a meaningful estimate of the exact location and number of qtl for that given trait. eight phs qtl on chromosomes 3a, 3b, 3d and 4a were identified in a meta-qtl study involving different populations. a high level of genome-synteny exists among wheat, brachypodium and rice, with wheat being more closely related to brachypodium than to rice. conservation or collinearity of genetic markers and greater structural similarities in the coding regions of orthologous genes of wheat and brachypodium have been reported. however, given differences in gene content in orthologous regions of wheat, brachypodium and rice, it might be beneficial to use both genomic sequences of brachypodium and rice in comparative mapping studies for map based cloning and gene discovery in wheat. our study deployed snp markers from a 90k infinium iselect custom beadchip, in addition to available ssr, dart and ests, to generate high density genetic maps for the identification of phs resistance qtl. sequences corresponding to polymorphic snps flanking phs qtl were analyzed against genomic sequences of brachypodium and rice. the objectives of our research were a) to identify candidate genes and regions in brachypodium and rice that are orthologous to phs resistance qtl intervals in wheat, and b) to utilize sequences of snps flanking phs qtl to develop kasp markers for mas of phs resistance.  linkage mapping a total of snp, ssr, dart and est markers were mapped to all wheat chromosomes. the resulting linkage map spanning  cm is reported in additional file of the markers, or % were snps, while the remaining or % comprised ssr, dart and est markers. the largest number of snp markers were distributed across the b genome, followed by snps mapped to the a genome, and snp markers on the d genome.table 1table cumulative map-lengths of a, b and d genome chromosomes alongside corresponding genome-wise distribution of snp markers mapped in the hexaploid dh population of rl4452/‘ac domain’ genome map length mapped markers snps ssrs, darts & ests % snps qtl analysis phs datasets were analyzed with both mim and simple interval mapping methods. as results of both methods were very similar, only those of mim were reported in this study. the mim analysis identified four qtl with significant effects, located across chromosomes 3b, 4a, 7b and 7d. each of these four qtl appeared in two or more environments and had peak lod scores greater than the critical threshold lod at 5% significance levels. coincident qtl for gi, si and fn were located on chromosome 4a. across trials, rl alleles on 3b and 7b provided phs resistance as they reduced si. however, ‘ac domain’ alleles also provided phs resistance as they increased fn on 4a and 7d and reduced si and gi on 4a.table results of multiple interval mapping: four qtl for phs traits identified on chromosomes 3b, 4a, 7b. and 7d. in a dh population of rl4452/‘ac domain’ replicated in multi-year environments qtl trial dataset chromosome qtl peak location additive a % pv lod α germination index qgi.crc-4a glenlea2005 qgi.crc-4a winnipeg2004 qgi.crc-4a winnipeg2005 sprouting index qsi.crc-3b glenlea2005 qsi.crc-3b winnipeg2004 qsi.crc-4a glenlea2005 qsi.crc-4a winnipeg2004 qsi.crc-4a winnipeg2005 qsi.crc-4a swift current2003 qsi.crc-7b swift current2003 qsi.crc-7b swift current2004 falling number qfn.crc-4a glenlea2005 qfn.crc-4a winnipeg2004 qfn.crc-7d glenlea2003 qfn.crc-7d glenlea2005 apositive or negative additive values relate to allele effects of the ac domain parent. candidate regions and genes for phs resistance sequences of snps flanking qtl for phs resistance on chromosomes 3b, 4a, 7b and 7d were subjected to blastn searches on the iwgsc and gramene databases and returned hits to candidate regions in brachypodium and rice. genetic and physical maps displaying orthologous regions for phs resistance in wheat, brachypodium and rice are given in figures 1a and b. a  cm qtl interval on chromosome 3b was orthologous to a ~  mb region on chromosome of brachypodium and to a ~  mb region on chromosome of rice. the 4a qtl interval was  cm and was orthologous to a ~  mb region on chromosome of brachypodium and to a ~  mb region on chromosome of rice. on chromosome 7b. the qtl interval spanned  cm and was orthologous to a ~  mb region on chromosome of brachypodium and to a ~1 mb region on chromosome of rice. the qtl interval on 7d. was  cm and was orthologous to a ~  mb region on chromosome of brachypodium, and a ~  mb region on chromosome of rice.table genetic map locations of snp markers flanking phs qtl on chromosomes 3b, 4a, 7b. and 7d. in a wheat dh population of a rl4452/‘ac domain’ cross and their corresponding physical locations/candidate regions in brachypodium distachyon and rice snp marker map survey sequence blastn hits to brachypodium genes blastn hits to rice genes contig no. chromosome 3b chromosome 4a chromosome 7b.1 chromosome 7d.2!weak hit to genomic regions in brachpodium or rice that is orthologous to the qtl interval for phs resistance in wheat. best hits that do not correspond to the candidate region in brachpodium or rice are in italics. location of qtl and syntenic regions in brachpodium and rice. a. location of qtl and flanking markers for phs resistance on a) wheat chromosome 3b and its candidate regions on brachypodium bd and rice os and b) chromosome 7b. and its candidate regions on brachypodium bd and rice os b. location of qtl and flanking markers for phs resistance on a) wheat chromosome 4a and its candidate regions on brachypodium bd and rice os and b) chromosome 7d. and its candidate regions on brachypodium bd and rice os brachypodium/rice candidates for phs resistance orthologous to consensus regions on wheat chromosomes 3b, 4a, 7b and 7d were identified. in the 3b region there are genes in the brachypodium orthologous region and in the rice region. the 4a region had genes in the brachypodium region and in rice, while the 7b region had in brachypodium and in rice and the 7d region had in brachypodium and in rice. genes involved in aba and ga metabolism as well as those affecting flowering time were present in the qtl regions. among these were bradi2g49795/os01g, os01g os01g orthologous to chromosome 3b, bradi1g00950/os03g, os03g os03g orthologous to 4a, bradi1g46060/os06g orthologous to 7b, bradi1g bradi1g bradi1g bradi1g, bradi1g bradi1g and bradi1g48830/os06g orthologous to chromosome 7d. development and validation of kasp primers a total of kasp markers, five each for chromosomes 3b and 7b. and four each for chromosomes 4a and 7d. were developed from sequences of snps flanking qtl for phs resistance. genetic map locations of individual kasp markers were identical to the respective snp from which they were derived. primer sets of all kasp markers are listed in additional file further, we validated the conversion of these kasps from matching genotype calls of infinium snp data on dh progeny genotypes. four dh progeny genotypes of the rl4452/‘ac domain’ cross were identified to carry phs resistance on chromosomes 3b, 4a, 7b and 7d.table a list of competitive allele-specific pcr markers developed for mas of phs from snps flanking phs qtl on chromosomes 3b, 4a, 7b and 7d in a dh population of a rl4452/‘ac domain’ cross sl. kasp marker source snp chr phs trait si si si si si gi, si, fn gi, si, fn gi, si, fn gi, si, fn si si si si si fn fn fn fn discussion the objectives of our research were to identify candidate regions for phs resistance qtl of wheat and develop kasp markers from sequences of snps flanking such qtl. this is an important step in the process of map-based cloning of genes that underlie important quantitative traits like phs resistance. our objectives were achieved using snps from the 90 k infinium custom beadchip to develop a high density linkage map in the rl4452/‘ac domain’ mapping population and subsequently detect qtl for phs resistance on chromosomes 3b, 4a, 7b and 7d. comparative mapping utilizing sequences of snps flanking phs resistance qtl enabled identification of candidate genes and regions in brachypodium and rice. the resulting kasp markers can be deployed in future genetic studies of phs, and in evaluation of phs in germplasm and breeding material. of the mapped markers, or % were snp markers, while the remaining or % were ssr, dart and est markers. the b genome chromosomes accounted for the largest number of snp markers, followed by the a genome with snps, and the d genome with snp markers. a likely explanation for larger numbers of b genome snp markers could be the greater genetic diversity of b genome species when compared to the a and d genome species. a faster rate of evolution of the b genome due to greater polymorphism and duplication events, in addition to greater genetic diversity brought about by cross pollination were cited as possible explanations for findings of a greater number of ests associated with more unique loci on the b genome when compared to the a and d genomes. phs datasets were analyzed with both mim and sim methods. because results of both methods were very similar, only those of the mim analyses were reported. as qtl identified using mim were robust and supported by sim results, it is unlikely that additional large effect qtl involved in epistatic interactions might have been detected using other qtl mapping methods that detect both main effect and epistatic qtl. further, a meta-qtl study reporting phs qtl on 4a and group chromosomes support significant phs qtl identified on chromosome 3b and 4a of our study. the most consistent of the four phs qtl identified on chromosomes 3b, 4a, 7b and 7d were located on chromosome 4a; gi, si and fn trait qtl each accounting for %, % and % of the phenotypic variation in their respective traits. the qtl for these phs traits were coincident and maybe associated with the same gene. these findings might suggest that chromosome 4a is involved in regulation of phs trait qtl in our test population. previous reports of the association of phs traits with chromosome 4a, support the importance of this qtl for phs in addition to a major si qtl on 4a, two other qtl for si were identified on chromosomes 3b and 7b. both si qtl on 3b and 7b. were detected in two of six environments. qtl that provide tolerance to late maturity α- amylase have been mapped on 3bs and 7bl in an australian wheat cross cranbrook/halberd. in both studies, the ssr markers xwmc xwmc xgwm xwmc xgwm xwmc xwmc and xgwm xwmc xwmc also flanked corresponding phs qtl intervals on chromosomes 3b. and 7b. respectively. further, alleles of a regulator gene vp-1b on 3b have been reported to influence grain dormancy in chinese wheat varieties. in a follow up study, the vp-1b locus was validated in a white-grained chinese landrace wanxianbaimaizi using ssr markers and a gene-specific primer vp a cim analysis identified a seed dormancy qtl qsd.ahau-3b on 3b flanked by vp which is linked to an ssr marker xwmc that also happens to flank the phs qtl interval on chromosome 3b of our study. the above findings suggest that phs and lma qtl on chromosomes 3b and 7b are likely the same. ‘ac domain’ alleles contributed to increasing the fn on 7d, with the exception of the glenlea trial, wherein a negative additive score was observed for the fn. while the fn qtl on chromosome 7d is unique to our study, a significant time to maturity qtl also on 7d, and a positive contribution of the rl allele, has been reported previously by in the same rl4452/‘ac domain’ population. the authors reported an ssr marker xgwm tightly linked to this qtl, which is distally located on 7dl, and is  cm from the qtl peak of our study. in the glenlea trial, the average fn score of dh progeny was the lowest of the four trials. the low fn score at this location might suggest greater levels of phs of ‘ac domain’ genotypes, probably brought on by wet weather conditions at the maturity stages or during the three weeks preceding harvest. as qtl locations of both these mat and fn traits nearly coincide and are influenced by negative and positive additive effects of ‘ac domain’ alleles respectively, the action of a pleiotrophic locus regulating both fn and mat could be assumed. at glenlea in it is possible that the lower fn for the domain allele is due to adverse weather conditions at maturity or that the 7d qtl identified here might not actually be a phs qtl, but rather a pleiotrophic effect of the mat qtl on phs. flanking marker intervals of a given phs trait qtl were not always the same across trials/datasets. it is quite likely that the respective underlying genes influencing each of these traits are the same; difference in qtl interval location being mainly due to environment or experimental error from differences in class means of individual trial data sets. alternatively, the possibility of two closely linked loci controlling the same trait cannot be ruled out. blastn searches with sequences of snp markers flanking phs qtl on chromosomes 3b, 4a, 7b and 7d revealed candidate regions in brachypodium and rice genomes. the qtl interval on chromosome 3b was orthologous to regions on bradi and the long arm of os while qtl intervals on chromosomes 4a were orthologous to regions on bradi and the short arm of os qtl intervals on chromosome 7b. and 7d. were orthologous to regions on bradi and the short arm of os of rice. the above findings of orthology between wheat/rice chromosomes: 3b/os 4a/os and 7b&7d/os concur with previous reports of wheat/rice chromosomal region similarities revealed via comparative mapping with dna probes and ests. further, orthologies between phs qtl intervals of 4a, 7b, 7d and genomic regions of bradi and 3b/bradi in our study will be refined to tease out individual genes responsible for variation in phs resistance. the availability of information on whole-genome assembled gene sequences of chinese spring and gene-orthologies among the said wheat and brachypodium chromosomes established using ests mapped to wheat deletion bins will serve as useful references to complement our efforts. eighteen kasp markers were developed from snp sequences flanking qtl for phs resistance. identical genotype calls of infinium snp data enabled validation of the kasp markers and identified four progeny genotypes of the rl4452/‘ac domain’ population possessing phs resistance on all four qtl on 3b, 4a, 7b and 7d. criteria for selection of these genotypes was based on findings of our study: ‘ac domain’ reduced gi and si on 4a, increased fn on 4a and 7d, while rl reduced si on chromosomes 3b and 7b. further, these kasp markers can be deployed in future genetic studies of phs, and in evaluation of phs in germplasm and breeding material. genes present in brachypodium and rice in orthologous regions corresponding to the qtl were identified. the 3b region is large and contains over genes in brachypodium and over in rice. more markers are needed to reduce the size of the region and the emerging reference sequence of chromosome 3b will be a valuable resource. there are a number of aba-inducible genes which could be a starting point to search for additional markers. the 4a and 7b regions contain many fewer genes in brachypodium and rice than the 3b region. gibberellin oxidase on bradi1/os orthologous to 4a and abscissic acid responsive elements on bradi1/os orthologous to chromosome 7b are candidates worth further study. ga - oxidase has previously been considered as a candidate gene underlying phs qtl on 4a. on chromosome 7d the qtl was coincident with a previously identified maturity qtl in the same population. genes affecting flowering time are present in the orthologous regions in brachypodium and rice. these include the far-red impaired responsive related proteins on chromosome bradi as well as vrn1-ap2/b3-like transcription factors on bradi and phosphatidylethanolamine - binding protein homologous to the flowering locus t gene on bradi1/os orthologous to chromosome 7d. because our study utilized a large number of sequence-based snps not available for previous mapping studies, the resulting genetic maps and qtl flanking snp markers are a novel and current resource for identification of underlying genes based on synteny and collinearity to model species brachypodium and rice. further, the identification of candidate genes and regions for phs in brachypodium and rice will enable a targeted focus for selection of candidate genes whose physiological/biological functions are linked to or influence variation in phs traits under study. such candidate gene-specific pcr markers will be developed and validated via mapping to the qtl intervals for phs resistance in wheat.  in our study we utilized snps from a wheat 90 k infinium iselect custom beadchip that permitted detection and assignment of significant phs resistance qtl to specific chromosomal locations on genetic maps. sequences of snps flanking phs resistance qtl enabled identification of candidate genes and regions for phs in brachypodium and rice via comparative mapping. the kasp markers resulting from this study can be suitably deployed in future genetic studies of phs and might also be useful in the evaluation of phs in germplasm and breeding material. 
this was achieved via high density mapping of single nucleotide polymorphism markers from an illumina 90 k infinium custom beadchip in a doubled haploid population derived from a rl4452/‘ac domain’ cross and subsequent detection of quantitative trait loci for phs related traits. our study identified candidate genes involved in abscissic acid and gibberellin metabolism, and flowering time in four genomic regions of brachypodium and rice respectively, in addition to kasp markers for phs resistance in wheat. a total of snp flanking sequences on chromosomes 3b, 4a, 7b and 7d were converted to kasp markers and validated with matching genotype calls of infinium snp data. the aim of this study was to identify candidate regions and develop molecular markers for phs resistance in wheat. these markers can be deployed in future genetic studies of phs resistance and might also be useful in the evaluation of phs in germplasm and breeding material. qtl analyses using multiple interval mapping identified four qtl for phs resistance traits on chromosomes 3b, 4a, 7b and 7d. best survey sequence hits were subject to a blastn search on gramene against both brachypodium and rice databases, and candidate genes and regions for phs resistance were identified. wheatpre-harvest sproutingquantitative trait locicandidate genesissue-copyright-statement© the author 2014 the availability of markers for marker-assisted selection of phs resistance will serve to enhance breeding selection and advancement of lines for cultivar development. snp marker sequences flanking qtl were used to locate colinear regions in brachypodium and rice, and identify genic markers associated with phs resistance that can be utilized for mas in wheat. a linkage map spanning  cm was constructed with a total of snp, simple sequence repeat, diversity arrays technology and expressed sequence tag markers.
 pre-harvest sprouting of wheat grain leads to a reduction in grain yield and quality. the availability of markers for marker-assisted selection of phs resistance will serve to enhance breeding selection and advancement of lines for cultivar development. the aim of this study was to identify candidate regions and develop molecular markers for phs resistance in wheat. this was achieved via high density mapping of single nucleotide polymorphism markers from an illumina 90 k infinium custom beadchip in a doubled haploid population derived from a rl4452/‘ac domain’ cross and subsequent detection of quantitative trait loci for phs related traits. snp marker sequences flanking qtl were used to locate colinear regions in brachypodium and rice, and identify genic markers associated with phs resistance that can be utilized for mas in wheat.  a linkage map spanning  cm was constructed with a total of snp, simple sequence repeat, diversity arrays technology and expressed sequence tag markers. qtl analyses using multiple interval mapping identified four qtl for phs resistance traits on chromosomes 3b, 4a, 7b and 7d. sequences of snps flanking these qtl were subject to a blastn search on the international wheat genome sequencing consortium database. best survey sequence hits were subject to a blastn search on gramene against both brachypodium and rice databases, and candidate genes and regions for phs resistance were identified. a total of snp flanking sequences on chromosomes 3b, 4a, 7b and 7d were converted to kasp markers and validated with matching genotype calls of infinium snp data.  our study identified candidate genes involved in abscissic acid and gibberellin metabolism, and flowering time in four genomic regions of brachypodium and rice respectively, in addition to kasp markers for phs resistance in wheat. these markers can be deployed in future genetic studies of phs resistance and might also be useful in the evaluation of phs in germplasm and breeding material. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords wheatpre-harvest sproutingquantitative trait locicandidate genesissue-copyright-statement© the author 2014 