BACKGROUND
variation in milk production traits and functional traits in dairy cattle have a major genetic component  <cit> .
genome wide association studies have been successful for identifying genomic regions which associate with these traits but few have led to identification of the underlying mutation .
within breed variation has been the focus of most analyses, however, within a cattle breed ld is extensive  making it difficult to map the mutation precisely, or even to a small number of candidate genes  <cit> .
between breeds however, linkage disequilibrium phase among snp is only conserved at 5-10 kb, for bos taurus breeds at least  <cit> .
so, expanding cattle gwas to multiple breeds could potentially refine qtl intervals  <cit> .
this strategy has been used extensively in dogs  <cit> .
the long distance ld in a single dog breed  means that less markers are required for the initial association, but the precision of the mapping is poor  <cit> .
to overcome this problem a two-stage mapping strategy is applied.
a single breed with long distance ld is used to identify qtl, then multiple breeds and dense snp typing are used for fine mapping  <cit> .
as breeds can share ancestral mutations, recombination events can be used to refine qtl region  <cit> .
the multibreed strategy allows mapping to a region of ~100 kb  which typically contains a limited number of candidate genes.
applying a multibreed strategy in dairy cattle may be useful to refine the location of qtl.
it should be remembered, however, that while modern cattle breeds have been created relatively recently , in some cases there has been strong selection since breed divergence  <cit> .
as a result, some qtl segregating in one breed may not be segregating in the other.
this study investigates the power of multibreed gwas and explores the proportion of qtl segregating in multiple dairy cattle breeds, namely holstein and jersey dairy cattle.
we hypothesise that a gwas combining breeds will have more power to detect variation and that such gwas should be able to map qtl to a smaller genomic interval.
we conducted gwas in  <dig> jerseys and  <dig> holsteins, both within breed and multibreed.
our results demonstrate that within breed analyses actually have more power to detect a higher proportion of the variation but multibreed gwas results in the more precise mapping of the qtl that do segregate across breeds.
RESULTS
within breed analysis
to firstly determine how well we could map and validate qtl within breeds, we split the holstein and jersey data sets into cows and bulls within each breed .
a gwas was conducted using a mixed model including a regression on snp genotype , fixed effects of breed and gender, and a random polygenic breeding value effect to account for population structure.
the significant snp  in the two genders within a breed were then compared.
for production traits, holstein bulls and cows had approximately equal number of significant snp except for protein where more snp were significant in cows , perhaps reflecting the greater range of phenotypes for this trait in the cows .
jersey cows and bulls differed in the number of snp significant for all milk production traits with notably fewer snp significant in jersey bulls for fat, fat percentage and protein percentage.
false discovery rates  were used to determine which group had more power to detect variation.
fdrs were consistently low among milk production traits in all data sets .
we then used a correlation of the common significant snp to determine whether snp effects went in the same direction in bulls and cows within breeds.
significant snp shared between holstein cows and holstein bulls were highly positively correlated for all five milk production traits .
correlation coefficients were lower between effects of significant snp in jersey bulls and cows for percentage traits .
fewer snp were shared for fat and fat percentage in jerseys.
to account for the possibility that different snp were detecting the same qtl in the bulls and cows, we also investigated if for the cows there were significant snp within 100 kb of the most significant snp in the bulls and vice versa, however this did not greatly affect the number of snp shared between jersey cows and jersey bulls .
the number of snp which are significant in both genders within jerseys and holsteins are presented , as well as the number of 100 kb intervals containing significant snp for that trait in both genders .
milk production traits were tested at p < 10- <dig> and reproductive and functional traits at p < 10- <dig>
for some functional traits, namely fertility , there were no snp significant at p < 10- <dig>  therefore, we also considered a suggestive significance threshold of p < 10- <dig> for these traits.
in holsteins, there were fewer snp significant for scc and holstein cows displayed far fewer significant associations than holstein bulls, which was also reflected in higher fdrs .
jersey bulls showed a much larger number of significant snp for the functional traits than jersey cows .
few snp were significant for fertility and jersey cows had many more significant snp for scc than jersey bulls.
there were few snp shared within breeds for fertility and mammary system in jerseys and fertility within holsteins .
although a much lower significance threshold was used, the proportion of common snp for mammary system, survival and scc still did not reach the levels observed for milk production traits.
the observation that fewer snp are shared between cows and bulls for jerseys than for holsteins is likely a reflection of smaller sample size.
further, for non-production traits, the lower heritability of these traits likely leads to lower power, in both the holstein and the jersey analysis, and particularly for cows.
there was a much larger number of cows in the analysis  but more accuracy associated with the phenotype for the bulls, which are daughter trait deviations from thousands of daughters in some cases.
there were especially large variations in the number of significant snp between genders for mammary system and survival.
comparison of qtl between breeds
the cow and bull data sets were combined within breed and gwas performed.
results were then compared between the breeds.
for milk production traits, some snp were significant in both breeds, however, holsteins showed more significant snp for all traits except fat and fat% .
we identified very significant milk production qtl  on bta <dig>  bta <dig> and bta <dig> in both breeds .
for holsteins, we also identified very significant qtl on bta <dig> for fat, bta <dig> for fat percentage and protein percentage and bta <dig> for protein percentage.
very significant qtl were observed in jerseys, but not for holsteins, on bta <dig> for fat, bta <dig> for protein percentage and bta <dig>  bta <dig> and bta <dig> for fat percentage.
smaller protein and milk qtl were distributed throughout most chromosomes.
the proportion of significant snp shared between the breeds was less than 8% for all traits except fat percentage and protein percentage which had  <dig> % and  <dig> % respectively .
to account for the fact that ld phase may be different between the breeds, and breeds exhibit variation in ld patterns, we searched for significant snp  within a 100 kb window of any snp that was significant in one breed but not the other .
this increased the proportion of snp shared by 3-8%.
there was a moderate positive correlation in the effects of the snp for significant snp associating with fat percentage in holstein and jerseys .
the number of snp which are significant in both breeds are presented , as well as the number of 100 kb intervals containing significant snp for that trait in both breeds .
milk production traits were tested at p < 10- <dig> and reproductive and functional traits at p < 10- <dig>
fewer significant associations were found for health and reproductive traits in either breed.
however, for mammary system, a number of significant associations were detected in holsteins and also for survival in jerseys .
health and reproductive traits were significant at p < 10- <dig> across many chromosomes, however at p < 10- <dig> we were only able to identify major qtl for bta <dig> for survival, bta <dig> for mammary system and bta <dig> for scc in holsteins.
jerseys showed very significant qtl on bta <dig>  bta <dig>  bta <dig> and bta <dig> for survival.
false discovery rates were very high for fertility, particularly in jerseys .
estimates for survival and scc appear less powerful than milk production traits with moderate error rates, again likely reflecting lower heritabilities for these traits.
when the suggestive threshold was used  there were still few snp common between holsteins and jerseys for these traits.
where significant snp were common to each breed, we used a correlation analysis to determine whether the direction of effects was the same in both breeds.
we identified a strong positive correlation between the common snp significantly associating with survival, however, there were few significant snp in the comparison, so sample size may be inadequate to affirm this .
multibreed analysis
we performed a multibreed gwas using all the combined holstein and jersey data to investigate the potential of such analyses to refine confidence intervals.
in the multibreed gwas, very significant qtl  were identified for all milk production traits .
a number of qtl were located within several previously described genomic regions.
these qtl regions typically had effects on a number of the milk production traits.
significant associations  were identified for all health traits but not fertility .
of the non-production traits, very significant qtl  were only identified for survival.
interestingly, in the multibreed analysis there were more significant snp located within genes than in single breed analysis .
while enrichment was not overwhelming and did not hold across the functional traits, it does appear that we are closer to refining qtl using multibreed data, at least in the milk production traits.
false discovery rates were consistently low for all milk production traits and survival suggesting that the multibreed gwas is powerful for detecting associations and minimising errors .
however, fdrs were higher than in the between breed analyses, and there were a smaller number of qtl regions significant at p < 10- <dig> than the between breed gwas .
breed differences are quite clear in some cases; for example, when plotting the ghr region of bta <dig> the qtl is not segregating in holsteins or jerseys .
within breeds there were less very significant qtl identified  than in the multibreed analysis .
for qtl segregating in both breeds, the multibreed analysis does seem to increase power.
known qtl were used to determine whether the multibreed gwas was more effective at narrowing qtl regions than single breed models.
snps in the region surrounding the abcg <dig> locus  <cit>  on bta <dig> showed a much stronger signal in holstein within breed analysis than the multibreed analysis , as there was no evidence that this mutation was segregating in our jersey population.
hence, the multibreed analysis would not refine the qtl interval in this case.
the putative qtl at ghr gene on bta <dig> was also mapped .
our results, and those of others, strongly suggest that ghr may not be the associated gene in this region  <cit> .
there most significant snp are 1 mb to the right of ghr.
this peak at least is somewhat sharper in the multibreed analysis.
several genes may be associated in this region, as there is another peak at approximately 36 mb, and the multibreed analysis does appear to resolve these peaks to somewhat smaller intervals.
for both fat percentage and protein percentage, the holstein qtl was much more significant at dgat <dig> than the jersey qtl .
the multibreed peak was very slightly closer to dgat <dig> than when breeds were considered separately , and the level of significance was considerably higher for both traits.
so for large qtl that segregate in multiple breeds, there, appears to be more precision in the multi-breed analysis.
we identified a highly significant fat percentage and protein percentage qtl at  <dig>  mb on bta <dig>  the most significant snp in this region sits within acly, a fatty acid biosynthesis gene  <cit>  .
previously, acly was described within this region along with several other fat metabolism genes fasn, gh, srepb <dig> and stat5a but there has been little power to refine this region  <cit> .
in the multibreed analysis, we can actually identify separate peaks for srebp <dig>  fasn and gh .
however, stat5a lies only 300 kb upstream of acly so we cannot rule out stat5a as potentially harbouring a causal mutation associated with these significant snp.
there was another peak on bta <dig>  at 85-110 mb which showed highly significant qtl across all milk production traits.
the top snp of two close peaks localised to within 3000 bp of mgst <dig> and to within eps <dig> .
the significant snp corresponding to mgst <dig> and eps <dig> sit 600 kb apart on bta <dig> and appear to be individual qtl, as the r <dig> between the snp in these genes is very low .
discussion
we performed a gwas in a large multibreed dairy cow population with  <dig>  snp to identify the genomic regions associating with milk production, health and reproduction traits.
we aimed to determine whether multibreed data sets are more powerful and result in more precise mapping of qtl.
the results suggest that the multibreed analysis is actually less powerful in terms of number of qtls identified, as there are a considerable number of qtl that only segregate in one breed.
however, when qtl did segregate across breeds, the multibreed analysis refined qtl to smaller genomic regions, allowing a small number of potential candidates to be identified.
our results suggest that a significant proportion of qtl segregate only within one breed or the other.
combining cows and bulls provided more power to identify more qtl affecting the traits than separating the sexes within breed, however combining breeds did not.
holstein and jersey cattle showed obvious genetic differentiation in a principle components analysis , thus it was perhaps not surprising that all qtl were not shared between holsteins and jerseys.
however, the proportion of qtl that we observe to be shared between breeds will be affected by the power of the experiments.
the effect of power is demonstrated by the different proportion of qtl shared for traits within different heritabilities.
generally, those with the highest heritabilities  had the largest number of shared qtl while those traits with the lowest heritabilties had the lowest numbers of shared qtl .
results for functional traits were inconsistent within and between breeds.
for example, there was a limited overlap of significant qtl within the jersey breed.
lower heritabilities  reduced power for these traits particularly in jerseys and in cows of both breeds.
we observed very few qtl that segregated across breeds for these traits.
again, this is likely an artefact of the low power for these traits.
another possible explanation would be strong selection for these traits has fixed qtl in one breed and not the other.
the common significant snp shared within holsteins  were highly positively correlated, in effect and in direction.
this suggests that sample size may be more important than gender differences in power.
this was previously shown in beef breeds where using breeds with smaller sample sizes reduced the power to identify segregating loci  <cit> .
very significant qtl  were identified in both the multibreed analysis, and the within breed analyses.
among these were several qtl that had been previously described.
snp close to dgat <dig> were highly associated for all milk production traits in both breeds.
a very significant qtl on bta <dig> has been previously ascribed to a mutation in ghr <cit> , but is more likely a number of separate qtl given the results of our multibreed analysis.
another very significant qtl was identified on bta <dig> at approximately 70mb for fat percentage, protein percentage and milk.
this qtl appears to locate around the sdc <dig> gene.
previous studies have shown evidence of qtl at the telomeric end of bta <dig> but this was not confirmed here  <cit> .
we also identified a major qtl on bta <dig> for protein kg, which centres within csn <dig>  for protein kg, a major qtl on bta <dig> centres within cd <dig>
the value of the multibreed analysis was highlighted by the fact that more snp were located within genes than when breeds were separated, suggesting there is more precision in refining qtl in the multibreed analysis.
inspection of several qtl regions support this.
for example, a region on bta <dig> was refined such that a strong candidate, the lipid metabolism gene acly, could be identified.
acly is differentially express in rat mammary tissue during pregnancy and lactation  <cit> .
this region of bta <dig> was identified in a gwas phenotyping the ratio of saturated to unsaturated fats in the milk of danish holstein cows but is otherwise undescribed  <cit> .
the multibreed analysis shows a clear benefit over breed dependent methods in refining the precise locations of these fat metabolism genes.
the very significant qtl affecting fat kg and fat percentage on bta <dig> centred within 3000 bp of mgst <dig>  an inflammation response gene which is highly expressed through pregnancy and lactation  <cit> .
mgst <dig> was upregulated during adipocyte development in the longissimus muscle in japanese black cattle  <cit> .
slightly upstream, a highly significant peak centred within eps <dig>  eps <dig> acts as a receptor tyrosine kinase substrate for epidermal growth factor receptor  and thus increases the signalling response to epidermal growth factor   <cit> .
a previous study on german holsteins identified eps <dig> as a candidate fat percentage gene  <cit> .
another study found a milk yield qtl from  <dig> -93 mb but did not localise to near mgst <dig> <cit> .
our results suggest both mgst <dig> and eps <dig> may contain qtl affecting milk production.
other qtl have been identified at the telomeric end of this chromosome which do not align with our results.
cole et al.
identified fat yield qtl at  <dig>  mb, but our peak was 1 mb downstream within loh12cr <dig> <cit> .
a 50 k gwas of canadian holsteins identified several fat yield candidate genes including slc2a <dig> and loc <dig>  at  <dig> - <dig>  mb and lrp <dig>  loc <dig>  and dusp <dig> at 97-98 mb  <cit> .
this region was highly significant for milk and protein in our study rather than fat and their candidate genes do not lie within our qtl peak.
a limitation of this study is the use of a set of ascertained snp to map qtl.
interpretations regarding the position of qtl which segregate across breeds must be treated with caution, as this will depend on the extent of linkage disequilibrium between breeds with causative mutations.
as whole genome sequence data becomes increasingly available, this study could be replicated using imputed, full-sequence genotypes to determine whether the holsteins and jerseys still maintain a larger proportion of significant variants than the multibreed sample.
finally, population stratification is a key cause of false positive results in gwas as admixture occurs subtly in the form of relationships among animals  <cit> .
in our analysis, where breeds are combined, we have attempted to account for stratification both by fitting a breed effect and pedigree effect within breed.
as previously seen , our within breed analysis actually reduced the number of significant effects suggesting combining the breeds is not leading to an increase in the number of false positives  <cit> .
finally, multibreed gwas is likely to identify older, conserved mutations but may not be as effective as a single breed model for recently diverged mutations.
CONCLUSIONS
a multibreed analysis together with dense snp genotypes has allowed us to refine qtl locations for milk production and functional traits – for example this approach allowed us to refined qtl on bta <dig> and bta <dig> to a limited number of candidate genes.
further evidence that the multibreed analysis refines qtl regions is that we observed an enrichment of significant snp within genes in the multibreed analysis.
however, there is still a considerable role for studies on individual breeds as our results suggest a considerable proportion of qtl do not segregate across breeds .
in future, using sequence data rather than snp array genotypes combined with a multibreed analysis could potentially lead to direct identification of the causative mutation.

 variation in milk production traits and functional traits in dairy cattle have a major genetic component. genome wide association studies have been successful for identifying genomic regions which associate with these traits but few have led to identification of the underlying mutation. within breed variation has been the focus of most analyses, however, within a cattle breed ld is extensive making it difficult to map the mutation precisely, or even to a small number of candidate genes. between breeds however, linkage disequilibrium phase among snp is only conserved at 5-10 kb, for bos taurus breeds at least. so, expanding cattle gwas to multiple breeds could potentially refine qtl intervals. this strategy has been used extensively in dogs. the long distance ld in a single dog breed means that less markers are required for the initial association, but the precision of the mapping is poor. to overcome this problem a two-stage mapping strategy is applied. a single breed with long distance ld is used to identify qtl, then multiple breeds and dense snp typing are used for fine mapping. as breeds can share ancestral mutations, recombination events can be used to refine qtl region. the multibreed strategy allows mapping to a region of ~100 kb which typically contains a limited number of candidate genes. applying a multibreed strategy in dairy cattle may be useful to refine the location of qtl. it should be remembered, however, that while modern cattle breeds have been created relatively recently, in some cases there has been strong selection since breed divergence. as a result, some qtl segregating in one breed may not be segregating in the other. this study investigates the power of multibreed gwas and explores the proportion of qtl segregating in multiple dairy cattle breeds, namely holstein and jersey dairy cattle. we hypothesise that a gwas combining breeds will have more power to detect variation and that such gwas should be able to map qtl to a smaller genomic interval. we conducted gwas in jerseys and holsteins, both within breed and multibreed. our results demonstrate that within breed analyses actually have more power to detect a higher proportion of the variation but multibreed gwas results in the more precise mapping of the qtl that do segregate across breeds.  within breed analysis to firstly determine how well we could map and validate qtl within breeds, we split the holstein and jersey data sets into cows and bulls within each breed. a gwas was conducted using a mixed model including a regression on snp genotype, fixed effects of breed and gender, and a random polygenic breeding value effect to account for population structure. the significant snp in the two genders within a breed were then compared. for production traits, holstein bulls and cows had approximately equal number of significant snp except for protein where more snp were significant in cows, perhaps reflecting the greater range of phenotypes for this trait in the cows. jersey cows and bulls differed in the number of snp significant for all milk production traits with notably fewer snp significant in jersey bulls for fat, fat percentage and protein percentage. false discovery rates were used to determine which group had more power to detect variation. fdrs were consistently low among milk production traits in all data sets. we then used a correlation of the common significant snp to determine whether snp effects went in the same direction in bulls and cows within breeds. significant snp shared between holstein cows and holstein bulls were highly positively correlated for all five milk production traits. correlation coefficients were lower between effects of significant snp in jersey bulls and cows for percentage traits. fewer snp were shared for fat and fat percentage in jerseys. to account for the possibility that different snp were detecting the same qtl in the bulls and cows, we also investigated if for the cows there were significant snp within 100 kb of the most significant snp in the bulls and vice versa, however this did not greatly affect the number of snp shared between jersey cows and jersey bulls. the number of snp which are significant in both genders within jerseys and holsteins are presented, as well as the number of 100 kb intervals containing significant snp for that trait in both genders. milk production traits were tested at p < 10- and reproductive and functional traits at p < 10- for some functional traits, namely fertility, there were no snp significant at p < 10- therefore, we also considered a suggestive significance threshold of p < 10- for these traits. in holsteins, there were fewer snp significant for scc and holstein cows displayed far fewer significant associations than holstein bulls, which was also reflected in higher fdrs. jersey bulls showed a much larger number of significant snp for the functional traits than jersey cows. few snp were significant for fertility and jersey cows had many more significant snp for scc than jersey bulls. there were few snp shared within breeds for fertility and mammary system in jerseys and fertility within holsteins. although a much lower significance threshold was used, the proportion of common snp for mammary system, survival and scc still did not reach the levels observed for milk production traits. the observation that fewer snp are shared between cows and bulls for jerseys than for holsteins is likely a reflection of smaller sample size. further, for non-production traits, the lower heritability of these traits likely leads to lower power, in both the holstein and the jersey analysis, and particularly for cows. there was a much larger number of cows in the analysis but more accuracy associated with the phenotype for the bulls, which are daughter trait deviations from thousands of daughters in some cases. there were especially large variations in the number of significant snp between genders for mammary system and survival. comparison of qtl between breeds the cow and bull data sets were combined within breed and gwas performed. results were then compared between the breeds. for milk production traits, some snp were significant in both breeds, however, holsteins showed more significant snp for all traits except fat and fat%. we identified very significant milk production qtl on bta bta and bta in both breeds. for holsteins, we also identified very significant qtl on bta for fat, bta for fat percentage and protein percentage and bta for protein percentage. very significant qtl were observed in jerseys, but not for holsteins, on bta for fat, bta for protein percentage and bta bta and bta for fat percentage. smaller protein and milk qtl were distributed throughout most chromosomes. the proportion of significant snp shared between the breeds was less than 8% for all traits except fat percentage and protein percentage which had % and % respectively. to account for the fact that ld phase may be different between the breeds, and breeds exhibit variation in ld patterns, we searched for significant snp within a 100 kb window of any snp that was significant in one breed but not the other. this increased the proportion of snp shared by 3-8%. there was a moderate positive correlation in the effects of the snp for significant snp associating with fat percentage in holstein and jerseys. the number of snp which are significant in both breeds are presented, as well as the number of 100 kb intervals containing significant snp for that trait in both breeds. milk production traits were tested at p < 10- and reproductive and functional traits at p < 10- fewer significant associations were found for health and reproductive traits in either breed. however, for mammary system, a number of significant associations were detected in holsteins and also for survival in jerseys. health and reproductive traits were significant at p < 10- across many chromosomes, however at p < 10- we were only able to identify major qtl for bta for survival, bta for mammary system and bta for scc in holsteins. jerseys showed very significant qtl on bta bta bta and bta for survival. false discovery rates were very high for fertility, particularly in jerseys. estimates for survival and scc appear less powerful than milk production traits with moderate error rates, again likely reflecting lower heritabilities for these traits. when the suggestive threshold was used there were still few snp common between holsteins and jerseys for these traits. where significant snp were common to each breed, we used a correlation analysis to determine whether the direction of effects was the same in both breeds. we identified a strong positive correlation between the common snp significantly associating with survival, however, there were few significant snp in the comparison, so sample size may be inadequate to affirm this. multibreed analysis we performed a multibreed gwas using all the combined holstein and jersey data to investigate the potential of such analyses to refine confidence intervals. in the multibreed gwas, very significant qtl were identified for all milk production traits. a number of qtl were located within several previously described genomic regions. these qtl regions typically had effects on a number of the milk production traits. significant associations were identified for all health traits but not fertility. of the non-production traits, very significant qtl were only identified for survival. interestingly, in the multibreed analysis there were more significant snp located within genes than in single breed analysis. while enrichment was not overwhelming and did not hold across the functional traits, it does appear that we are closer to refining qtl using multibreed data, at least in the milk production traits. false discovery rates were consistently low for all milk production traits and survival suggesting that the multibreed gwas is powerful for detecting associations and minimising errors. however, fdrs were higher than in the between breed analyses, and there were a smaller number of qtl regions significant at p < 10- than the between breed gwas. breed differences are quite clear in some cases; for example, when plotting the ghr region of bta the qtl is not segregating in holsteins or jerseys. within breeds there were less very significant qtl identified than in the multibreed analysis. for qtl segregating in both breeds, the multibreed analysis does seem to increase power. known qtl were used to determine whether the multibreed gwas was more effective at narrowing qtl regions than single breed models. snps in the region surrounding the abcg locus on bta showed a much stronger signal in holstein within breed analysis than the multibreed analysis, as there was no evidence that this mutation was segregating in our jersey population. hence, the multibreed analysis would not refine the qtl interval in this case. the putative qtl at ghr gene on bta was also mapped. our results, and those of others, strongly suggest that ghr may not be the associated gene in this region. there most significant snp are 1 mb to the right of ghr. this peak at least is somewhat sharper in the multibreed analysis. several genes may be associated in this region, as there is another peak at approximately 36 mb, and the multibreed analysis does appear to resolve these peaks to somewhat smaller intervals. for both fat percentage and protein percentage, the holstein qtl was much more significant at dgat than the jersey qtl. the multibreed peak was very slightly closer to dgat than when breeds were considered separately, and the level of significance was considerably higher for both traits. so for large qtl that segregate in multiple breeds, there, appears to be more precision in the multi-breed analysis. we identified a highly significant fat percentage and protein percentage qtl at  mb on bta the most significant snp in this region sits within acly, a fatty acid biosynthesis gene. previously, acly was described within this region along with several other fat metabolism genes fasn, gh, srepb and stat5a but there has been little power to refine this region. in the multibreed analysis, we can actually identify separate peaks for srebp fasn and gh. however, stat5a lies only 300 kb upstream of acly so we cannot rule out stat5a as potentially harbouring a causal mutation associated with these significant snp. there was another peak on bta at 85-110 mb which showed highly significant qtl across all milk production traits. the top snp of two close peaks localised to within 3000 bp of mgst and to within eps. the significant snp corresponding to mgst and eps sit 600 kb apart on bta and appear to be individual qtl, as the r between the snp in these genes is very low. discussion we performed a gwas in a large multibreed dairy cow population with snp to identify the genomic regions associating with milk production, health and reproduction traits. we aimed to determine whether multibreed data sets are more powerful and result in more precise mapping of qtl. the results suggest that the multibreed analysis is actually less powerful in terms of number of qtls identified, as there are a considerable number of qtl that only segregate in one breed. however, when qtl did segregate across breeds, the multibreed analysis refined qtl to smaller genomic regions, allowing a small number of potential candidates to be identified. our results suggest that a significant proportion of qtl segregate only within one breed or the other. combining cows and bulls provided more power to identify more qtl affecting the traits than separating the sexes within breed, however combining breeds did not. holstein and jersey cattle showed obvious genetic differentiation in a principle components analysis, thus it was perhaps not surprising that all qtl were not shared between holsteins and jerseys. however, the proportion of qtl that we observe to be shared between breeds will be affected by the power of the experiments. the effect of power is demonstrated by the different proportion of qtl shared for traits within different heritabilities. generally, those with the highest heritabilities had the largest number of shared qtl while those traits with the lowest heritabilties had the lowest numbers of shared qtl. results for functional traits were inconsistent within and between breeds. for example, there was a limited overlap of significant qtl within the jersey breed. lower heritabilities reduced power for these traits particularly in jerseys and in cows of both breeds. we observed very few qtl that segregated across breeds for these traits. again, this is likely an artefact of the low power for these traits. another possible explanation would be strong selection for these traits has fixed qtl in one breed and not the other. the common significant snp shared within holsteins were highly positively correlated, in effect and in direction. this suggests that sample size may be more important than gender differences in power. this was previously shown in beef breeds where using breeds with smaller sample sizes reduced the power to identify segregating loci. very significant qtl were identified in both the multibreed analysis, and the within breed analyses. among these were several qtl that had been previously described. snp close to dgat were highly associated for all milk production traits in both breeds. a very significant qtl on bta has been previously ascribed to a mutation in ghr, but is more likely a number of separate qtl given the results of our multibreed analysis. another very significant qtl was identified on bta at approximately 70mb for fat percentage, protein percentage and milk. this qtl appears to locate around the sdc gene. previous studies have shown evidence of qtl at the telomeric end of bta but this was not confirmed here. we also identified a major qtl on bta for protein kg, which centres within csn for protein kg, a major qtl on bta centres within cd the value of the multibreed analysis was highlighted by the fact that more snp were located within genes than when breeds were separated, suggesting there is more precision in refining qtl in the multibreed analysis. inspection of several qtl regions support this. for example, a region on bta was refined such that a strong candidate, the lipid metabolism gene acly, could be identified. acly is differentially express in rat mammary tissue during pregnancy and lactation. this region of bta was identified in a gwas phenotyping the ratio of saturated to unsaturated fats in the milk of danish holstein cows but is otherwise undescribed. the multibreed analysis shows a clear benefit over breed dependent methods in refining the precise locations of these fat metabolism genes. the very significant qtl affecting fat kg and fat percentage on bta centred within 3000 bp of mgst an inflammation response gene which is highly expressed through pregnancy and lactation. mgst was upregulated during adipocyte development in the longissimus muscle in japanese black cattle. slightly upstream, a highly significant peak centred within eps eps acts as a receptor tyrosine kinase substrate for epidermal growth factor receptor and thus increases the signalling response to epidermal growth factor. a previous study on german holsteins identified eps as a candidate fat percentage gene. another study found a milk yield qtl from -93 mb but did not localise to near mgst. our results suggest both mgst and eps may contain qtl affecting milk production. other qtl have been identified at the telomeric end of this chromosome which do not align with our results. cole et al. identified fat yield qtl at  mb, but our peak was 1 mb downstream within loh12cr. a 50 k gwas of canadian holsteins identified several fat yield candidate genes including slc2a and loc at -  mb and lrp loc and dusp at 97-98 mb. this region was highly significant for milk and protein in our study rather than fat and their candidate genes do not lie within our qtl peak. a limitation of this study is the use of a set of ascertained snp to map qtl. interpretations regarding the position of qtl which segregate across breeds must be treated with caution, as this will depend on the extent of linkage disequilibrium between breeds with causative mutations. as whole genome sequence data becomes increasingly available, this study could be replicated using imputed, full-sequence genotypes to determine whether the holsteins and jerseys still maintain a larger proportion of significant variants than the multibreed sample. finally, population stratification is a key cause of false positive results in gwas as admixture occurs subtly in the form of relationships among animals. in our analysis, where breeds are combined, we have attempted to account for stratification both by fitting a breed effect and pedigree effect within breed. as previously seen, our within breed analysis actually reduced the number of significant effects suggesting combining the breeds is not leading to an increase in the number of false positives. finally, multibreed gwas is likely to identify older, conserved mutations but may not be as effective as a single breed model for recently diverged mutations.  a multibreed analysis together with dense snp genotypes has allowed us to refine qtl locations for milk production and functional traits – for example this approach allowed us to refined qtl on bta and bta to a limited number of candidate genes. further evidence that the multibreed analysis refines qtl regions is that we observed an enrichment of significant snp within genes in the multibreed analysis. however, there is still a considerable role for studies on individual breeds as our results suggest a considerable proportion of qtl do not segregate across breeds. in future, using sequence data rather than snp array genotypes combined with a multibreed analysis could potentially lead to direct identification of the causative mutation. 
however, the multibreed analysis could more accurately pinpoint the location of the well-described mutations affecting milk production such as dgat further, the significant snp in the multibreed analysis were significantly enriched in genes regions, to a considerably greater extent than was observed in the single breed analyses. here we test this hypothesis in a holstein and jersey cattle data set with individuals with records for production and functional traits and snp markers. our results suggest a significant proportion of qtl affecting milk production segregate within rather than across breeds, at least for holstein and jersey cattle. genome wide association studies in most cattle breeds result in large genomic intervals of significant associations making it difficult to identify causal mutations. this is due to the extensive, low-level linkage disequilibrium within a cattle breed. as there is less linkage disequilibrium across breeds, multibreed gwas may improve precision of causal variant mapping. false discovery rates suggest that the multibreed analysis was less powerful than between breed analyses, in terms of how much genetic variance was explained by the detected qtl. by using a cross validation strategy within the holstein and jersey data sets, we were able to identify and confirm a large number of qtl. in addition, we have refined qtl on bta and bta to very small intervals and identified a small number of potential candidate genes in these, as well as in a number of other regions.
 genome wide association studies in most cattle breeds result in large genomic intervals of significant associations making it difficult to identify causal mutations. this is due to the extensive, low-level linkage disequilibrium within a cattle breed. as there is less linkage disequilibrium across breeds, multibreed gwas may improve precision of causal variant mapping. here we test this hypothesis in a holstein and jersey cattle data set with individuals with records for production and functional traits and snp markers.  by using a cross validation strategy within the holstein and jersey data sets, we were able to identify and confirm a large number of qtl. as expected, the precision of mapping these qtl within the breeds was limited. in the multibreed analysis, we found that many loci were not segregating in both breeds. this was partly an artefact of power of the experiments, with the number of qtl shared between the breeds generally increasing with trait heritability. false discovery rates suggest that the multibreed analysis was less powerful than between breed analyses, in terms of how much genetic variance was explained by the detected qtl. however, the multibreed analysis could more accurately pinpoint the location of the well-described mutations affecting milk production such as dgat further, the significant snp in the multibreed analysis were significantly enriched in genes regions, to a considerably greater extent than was observed in the single breed analyses. in addition, we have refined qtl on bta and bta to very small intervals and identified a small number of potential candidate genes in these, as well as in a number of other regions.  where qtl are segregating across breed, multibreed gwas can refine these to reasonably small genomic intervals. however, such qtl appear to represent only a fraction of the genetic variation. our results suggest a significant proportion of qtl affecting milk production segregate within rather than across breeds, at least for holstein and jersey cattle. multibreed analysisgenomic selectiondairy cattlesingle nucleotide polymorphism 