BACKGROUND
dairy cattle have been subjected to more than 60 years of intense selection for traits to enhance milk production .
relatively few bulls were chosen to produce thousands of daughters, resulting in large half-sib families.
traditionally, cattle breeders estimate the inbreeding coefficient by the degree of parental relatedness using pedigree and genotype data .
the rate of inbreeding in cattle populations has increased in recent years  <cit> , and there is a strong correlation between inbreeding levels and reduced fitness  <cit> .
this can be explained by the increased risk of homozygosity for deleterious alleles as inbreeding increases  <cit> .
thus, high levels of inbreeding in populations will result in inbreeding depression  <cit> .
reduced variability also leads to a reduced selection response in breeding programs  <cit> .
thus, maintaining genetic diversity is crucial in cattle breeding populations.
the availability of high-throughput whole genome sequencing for substantial numbers of animals has opened new avenues in examining genetic diversity and led to reliable and detailed investigation of large chromosome segments, including stretches of homozygous genomic regions  <cit> .
runs of homozygosity  are contiguous homozygous stretches in an individual genome due to transmission of identical haplotypes from parents to offspring.
roh detection can be used to improve mating systems and minimize inbreeding.
however, the effects of inbreeding vary among individuals and populations, and it has long been of interest to explore the mechanisms of inbreeding depression and deleterious variants at the genomic level .
recently, roh extracted from snp chip data have been used to study the population history of different cattle breeds  <cit> .
purfield et al.
<cit>  claimed that both natural and artificial selection of cattle, as well as demographic processes, have resulted in breeds with extensive phenotype variation.
roh may provide useful information on how these processes work in disparate populations, especially for cattle due to recent intense selection of sires, artificial insemination, and embryo transfer in some cattle breeds  <cit> .
however, roh estimated from snp chip data may miss short and medium roh due to limited resolution.
additionally, snp arrays suffer from ascertainment biases due to inclusion of snps with high minor allele frequencies  <cit> .
consanguineous mating, population size reduction, and selection result in long homozygous regions along the genome  <cit> .
two copies of a genome segment in an individual inherited from a common ancestor without recombination are identical-by-descent .
roh that arise due to inbreeding tend to be fairly long and are dispersed more or less randomly throughout the genome.
accumulation of deleterious variants by definition creates fitness consequences, particularly when homozygous  <cit> .
distribution of functional homozygote variants provides information on enrichment of deleterious homozygotes within homozygous regions compared with neutral homozygotes.
charlesworth et al.
<cit>  suggested that a large number of weakly deleterious variants are purged by negative selection.
a human genome study  <cit>  indicated that purifying selection interacted with founder effects during demographic processes, affecting the proportion of recessive deleterious variants.
recently, based on variant annotations, szpiech et al.
<cit>  reported long roh enriched deleterious variation in the human genome.
by counting deleterious and neutral variants inside and outside roh in humans, they determined that out of the two competing hypotheses regarding patterns of deleterious variation in the human genome — namely, whether a smaller or larger proportion of all deleterious homozygotes resided in roh regions compared with neutral homozygotes  <cit> , a larger proportion of deleterious homozygote variants were found, and deleterious variants in long roh accumulated more rapidly compared to short and medium roh in the human genome.
cattle domestication was initiated approximately  <dig>  years ago, with evidence of at least two separate domestication events  <cit> .
during the last few decades, breeders in modern dairy cattle breeding programs have implemented strong artificial selection.
kim et al.
showed that selection increases overall autozygosity across the genome, whereas the autozygosity in an unselected line does not change significantly across most chromosomes in cattle populations  <cit> .
genomic regions under positive selection show increased roh levels due to local reductions in haplotype diversity, i.e.
selective sweeps  <cit> .
certain haplotypes under these conditions constitute a large proportion of the total haplotype pool for the portions of the genome targeted for selective sweeps.
in these conditions, a portion of the roh is the actual selection target and is retained in the roh region  <cit> .
these roh will increase in frequency and be subject to purging, as the spread of haplotypes carrying deleterious recessive alleles are restrained by forces counteracting the selection pressure driving the selective sweep  <cit> .
therefore, we expect that this roh type would be on average shorter, composed of more common haplotypes, be concentrated in fewer genomic locations, and contain fewer deleterious alleles than the genomic average.
short roh are generally due to older haplotype relatedness, while longer roh result from more recent parental relatedness  <cit> .
thus, short and medium sized roh have been subject to selection for a longer period of time than longer roh.
furthermore, recombination will have had more time to trim down roh that have been the target of selective sweeps  <cit> .
roh regions are expected to exhibit an increased frequency of homozygotes compared to non-roh regions, as the homozygote allelic frequency in non-roh regions is p <dig> and the allelic frequency in roh regions is p, given a population allelic frequency of p. therefore, ibd results in enrichment of homozygotes in roh.
given the nature of cattle breeding, artificial selection is expected to play a more crucial role in shaping the frequency and distribution of functional variants in roh in modern cattle relative to human populations.
therefore, we expect that selection pressures could reduce the number of regions with an increased frequency of deleterious variations, and, at the same time, enrich for short roh regions with substantial beneficial effects in cattle breeding populations.
this remains an ongoing process, as the properties of genomic variants within roh regions continue to be discovered in cattle .
an increase in functional variant frequency in different roh length classes should also be examined under different population-genetic processes, such as the number of generations of inbreeding  <cit> .
genome scale bioinformatics annotations are available from a number of sources, including the variants effect predictor from ensembl  <cit> , with several available levels of annotation.
generally, synonymous polymorphisms are the least subject to selective forces as they have the least effect on the resultant protein.
tools such as sift can be used to predict non-synonymous change effects on proteins, and, thus, give an idea of their likely selective pressure  <cit> .
sift classifies non-synonymous changes as either non-deleterious or deleterious, based on the predicted effect on the protein.
by comparing these classes , we can study how selection has shaped deleterious variants within the cattle genomic roh.
we hypothesize that due to strong artificial selection pressures and demographic processes in cattle, deleterious variants increased in frequency within roh compared with non-deleterious variants and that the deleterious allelic frequency and distribution in roh classes differs markedly from the human genome.
testing this hypothesis will contribute to our understanding of inbreeding in cattle and help elucidate how artificial selection and other population level processes affect the distribution of functional variants.
to accomplish this, we examined patterns of roh detected using full genome sequencing in four danish cattle breeds and studied the distribution and frequency of deleterious and non-deleterious variations in different length roh regions.
RESULTS
general statistics
runs of homozygosity  in the autosomes of  <dig> resequenced individuals were determined from four danish dairy cattle breeds: holstein , jersey , old red danish dairy cattle , and new danish red dairy cattle  .
the average genomic roh content was  <dig>  % across the four cattle breeds, with hol, jer, new-red, and old-red having  <dig>  %,  <dig>  %,  <dig>  %, and  <dig>  %, respectively.
the average number of roh per genome was  <dig>  ±  <dig> , with an average size of  <dig> .2 bp, ranging from  <dig> kbp  to  <dig>  kbp .
the mean roh size varied significantly between hol, jer, old-red, and new-red  with the exception of jer and old-red .
the mean number of roh was significantly different between hol, jer, old-red, and new-red cattle  with the exception of hol and old-red .fig.
<dig> roh general statistics.
a average genome-wide nucleotide diversity ; b average nucleotide diversity outside roh ; c average roh size ; d average genome-wide roh totals
the average genome-wide nucleotide diversity  was  <dig>   heterozygous positions per kbp across all individuals, including roh, and  <dig>   heterozygous positions per kbp across all individuals in the genome excluding roh  .
the minimum and maximum nucleotide diversities were  <dig>   snps/kbp in jer and  <dig>   snps/kbp in new-red, respectively  and were significantly different between all cattle breeds except hol and new-red .
π-out was significantly different between old-red and hol, jer, and new-red  .
nucleotide diversity was higher in the vicinity of the major histocompatibility complex  on chromosome  <dig> .
genomic patterns of homozygosity
roh were separated into three size classes: small , medium , and large  .
the proportion of roh in each size class was computed in all  <dig> sequenced individuals.
while small roh were frequent throughout the genome, they constituted a small proportion of the entire genome .
in contrast, medium roh were much less frequent, they constituted significantly more of the genome than either small or large roh.
large roh were at least tenfold less numerous than medium roh, but nevertheless covered a sizable proportion of the total genome length.
old-red cattle on average had the largest proportion of their genome in large roh.
new-red cattle had fewer genomic roh and a smaller genomic proportion of roh than hol and jer cattle .
old-red and jer cattle on average had more roh and increased proportion of genomic homozygosity.fig  <dig> total roh number and genome proportions.
a the average small , medium , and large  roh numbers for the four breeds; b average total genome roh coverage for a given size class within each breed
principal component analysis successfully differentiated the four cattle breed individuals into different clusters based on ssr sequence data , with jer the most distantly related based on pca results.
three-dimensional plots did not show clear separation of cattle breeds based on to roh size, roh number, and π-out .
new-red cattle represented the most variable cluster due to high nucleotide diversity and fewer roh  .
there was lower nucleotide diversity and more total roh in jer compared to hol and new-red  .
despite its most distant origin, jer had lower nucleotide diversity , creating several clusters in the three dimensional plot .
however, all danish cattle breeds were more or less clustered together, with the exception of two new-red individuals .
based on sequence data, new-red cattle exhibited the fewest roh and smallest roh sizes.
this is a composite breed with contributions from other red breeds, including swedish red, finnish ayrshire, and brown swiss.
compared with new-red, these data suggest that the old-red breed has been more inbred based on relatively high coverage of long regions of homozygosity  , probably due to a relatively small breeding population and recent years of close mating.
furthermore, the sharing of roh regions was examined among sequenced individuals .
sharing of roh regions primarily happened in short rather than long roh regions, likely a result of combination of inbreeding and selection.
significant correlations were observed between fst, ihs, and shared roh regions in bins of 500 kb compared with the whole genome average  .
we also observed that instead of randomly distributed over the genomes , there were several obvious roh-dense peaks distributed shared between individuals across genomes .
therefore, the distribution of roh is not only result of pure demography, but likely the result of selection.
distribution of functional variants in roh regions
number of deleterious homozygous genotypes in roh
additional file 11: table s <dig> and additional file 12: table s <dig> show the counts for reference homozygotes , heterozygotes , and non-reference homozygotes  at deleterious and non-deleterious sites, respectively, that were contained within roh and non-roh regions .
the number of deleterious non-reference homozygotes was consistent with the roh coverage in all four breeds.
old-red and jer showed increased roh coverage, with a higher number of non-reference deleterious homozygotes in the genome.
non-reference non-deleterious homozygotes also exhibited the same trends as deleterious homozygotes.
figure  <dig> shows the total number of deleterious non-reference homozygotes  as a function of the total proportion of the genome covered by roh  for all sequenced individuals.
as roh coverage increased , a greater number of homozygotes were observed within roh, which was consistent with findings from the human genome  <cit> .
there was a very strong positive correlation between the number of deleterious homozygotes and the genomic roh proportion .
similarly, the number of homozygotes outside of roh decreased with the genomic roh proportion due to smaller non-roh regions as roh coverage increased.
as expected, there was a weak negative correlation between deleterious homozygotes outside roh and the genomic roh proportions .
compared with data from the human genome, the decreased slope for non-roh regions was much shallower than the increased slope for roh regions  <cit> .
however, this indicates that the increased deleterious homozygotes in roh regions exceed deleterious homozygote declines in non-roh regions in cattle.
similar to the human genome  <cit> , the fitted lines also predict that, on average, individual non-inbred cattle  carry approximately  <dig> deleterious homozygous variants.
an increased in roh coverage by 10 % will increase the expected deleterious homozygote numbers in roh regions by  <dig> and decrease the expected number of deleterious homozygotes in non-roh regions by  <dig>  yielding an expected net increase of  <dig> deleterious homozygotes.fig.
<dig> deleterious non-reference homozygotes versus the genome roh coverage in each individual.
red points represent the number of deleterious homozygotes falling within roh regions and black points represent the number of deleterious homozygotes falling outside roh regions
deleterious and non-deleterious homozygotes in roh of any size
figure 4a shows the proportion of deleterious non-reference homozygotes inside and outside roh regions  versus total genomic roh coverage .
the proportions of non-deleterious and deleterious homozygous genotypes within roh were strongly positively correlated with total genomic roh coverage .
these high correlations were expected, because as larger proportions of homozygous genotypes occur, roh coverage in the genome increases, and therefore roh comprise an increasingly greater proportion of the genome  <cit> .
the fi,rd proportion in genome-wide deleterious homozygotes within roh consistently exceeded the fi,rn proportion of genome-wide non-deleterious homozygotes within roh and the increasing slopes differed between deleterious and non-deleterious variants.fig.
<dig> the proportion of all genome-wide non-reference homozygotes falling in roh regions versus the genome roh coverage for each individual.
a any roh region; b short; c medium; and d long roh regions.
red points represent deleterious homozygotes, and blue points represent non-deleterious homozygotes
after fitting the two linear regression models, we found β <dig> was significant  indicating that the interaction between the two regression slopes  was significant.
however, β <dig> was not significant  between the two regression intercepts.
this is consistent with previous findings in humans  <cit> , where deleterious homozygotes showed increased frequency in roh relative to non-deleterious homozygotes and regression slopes were significantly different between deleterious and non-deleterious homozygotes.
deleterious and non-deleterious homozygotes by roh size
figure 4b shows fi,sd and fi,sn versus total genomic coverage for small roh  .
the proportion of non-deleterious homozygous genotypes in small roh, and the proportion of deleterious homozygous genotypes in small roh were positively correlated with genomic coverage .
figure 4c and d showed fi,md and fi,mn versus total genomic coverage for medium  and large  roh.
the regressions for homozygote numbers in medium  and large  roh had smaller p-values than in small roh.
these results show that deleterious homozygotes occur more frequently in roh than non-deleterious homozygotes.
additionally, when the proportion of deleterious homozygotes within large roh  is compared to the proportion within small roh , there was a substantially higher proportion of genome-wide deleterious homozygotes in small and medium vs. large roh especially in individuals with moderate to high roh coverage proportions .
given that roh coverage  for all individuals across the four breeds differed .
therefore, statistical tests for each size group were robust across the breeds and there were different roh coverage groups across all individuals .
similar trends were observed for each roh size group, and significantly different degrees of enrichment were observed within each size group.fig.
<dig> the genome-wide proportion of all non-reference homozygotes falling in different roh sizes versus genome roh coverage for each individual.
red, orange, and black points represent deleterious homozygotes in large, medium, and small roh regions, respectively
the intercepts and slopes of deleterious homozygotes and non-deleterious homozygotes were significantly different for large and medium roh , but slopes and intercepts were not significantly different between small and medium roh .
these results indicate inbreeding that generates short and medium roh increases the proportions of deleterious and non-deleterious homozygotes in roh regions compared to long roh.
if deleterious, non-deleterious, and synonymous homozygotes are considered as three separate classes, patterns similar to those observed in the deleterious and non-deleterious homozygotes analysis emerge.
deleterious homozygotes were at highest proportions in short and medium length roh.
there were smaller proportions of synonymous and non-deleterious homozygotes as genomic roh coverage of small and medium roh increases .
in large roh, the synonymous homozygote proportion over all homozygotes was higher than deleterious and non-deleterious homozygote proportions.
nonsense variants and roh
additional file 13: table s <dig> and additional file 14: table s <dig> report nonsense and loss of function nonsense sites, respectively, with counts for reference homozygotes , heterozygotes , and non-reference homozygotes , which fell into roh and non-roh regions.
figure 6a shows nonsense mutation distribution across all roh.
for low-roh individuals, the mean proportion of non-deleterious homozygote variants falling in roh marginally exceeded the nonsense or loss of function variants.
for high-roh individuals, however, the proportion of non-deleterious homozygotes within roh was lower than for nonsense homozygotes.
when roh are segregated by size , including individuals with high genomic roh coverage, the proportion of nonsense homozygotes in medium roh was greater than that of non-deleterious homozygotes, while the proportion of nonsense homozygotes was slightly lower than non-deleterious homozygotes for large roh.
this is consistent with the finding that high-roh individuals exhibited an increased proportion of damaging homozygotes  in roh of any size , primarily driven by medium roh .fig.
<dig> the proportion of all genome-wide non-reference homozygotes falling in roh regions for non-deleterious variants, nonsense variants, and loss of function nonsense variants versus the genome roh coverage for individuals in the “low roh” and “high roh” groups.
a: any roh region; b: short; c: medium; and d: long roh regions
discussion
cattle genomic roh patterns
the  <dig> individuals re-sequenced in the present study are key ancestors from the four danish cattle breeds.
the new-red is a composite breed with its primary origins in the old danish red, and includes contributions from other red breeds, including swedish red finnish ayrshire, and brown swiss cattle  <cit> .
our results showed roh size ranged from tens of kb to several mb and varied among individuals from different cattle breeds.
overall, medium sized roh were most common.
the average proportion of the genome represented by roh was  <dig>  % across all sequenced individuals.
however, pedigree records and lower density snp chips underestimated the inbreeding coefficient for these  <dig> bulls compared with that generated using roh from genome sequencing.
a previous study in danish cattle also reported a less than 5 % inbreeding coefficient using pedigree information  <cit> .
among breeds, jer and old-red were relatively more inbred compared with hol based on high roh genome coverage.
meanwhile, a high number and proportion of long roh were detected in old-red, likely indicating a small population with close mating for some period of time.
however, the smallest proportion of roh was detected in new-red, presumably due to its outbred origins.
these roh patterns are consistent with the known population history for these four breeds  <cit> .
pemberton et al.
allocated humans from different demographics to their corresponding place of origin by inferring population history from roh analysis  <cit> .
in our study, we also determined that individuals with similar roh distributions clustered together due to similar patterns of variation .
in contrast, pca generated different cluster types in the same sets of individuals based on population structure .
the fact that nucleotide diversity outside roh was higher in old-red than hol, jer, and new-red indicates that the historic genetic diversity of old-red might be relatively higher than the other breeds examined due to larger breeding populations for each of several past generations.
furthermore, evidence suggested old-red was the source population of new-red.
although the newly derived red populations exhibited the highest nucleotide diversity across the entire genome, the ancient haplotypes were more diverse in the ancestral old-red population.
reduced nucleotide diversity outside roh in new-red may also be explained by gene flow from hol to new-red.
in addition, nucleotide diversity levels outside roh presumably reflected the different origin of old-red from hol and jer.
bovine major histocompatibility complex  regions have long been known in the cattle genome  <cit> .
our analysis also detected mhc regions, which have a high degree of nucleotide diversity, on chromosome  <dig> .
pemberton et al.
reported a correlation between linkage disequilibrium levels and roh distribution in the human genome  <cit> .
mhc regions contain recombination hotspots  <cit>  and, therefore, high recombination rates to maintain relatively high levels of genetic diversity, but are subject to over dominant selection  <cit> .
roh are rarely present in mhc regions of the cattle genome, preventing random distribution of roh .
purfield et al.
<cit>  showed roh patterns in cattle populations using snp chip data.
our analysis is the first to use next-generation sequencing data to infer roh in cattle and indicate that long roh can only be detected with 50 k or hd snp chip data , while short roh are not detectable due to the required density of snp chip data .
short roh regions shared between individuals in our data  confirm that short roh were selected and derived from ancient haplotypes that became fixed in populations , while long roh are the result of more recent inbreeding events.
consequently, snp chip data misses information more relevant to historic inbreeding practices rather than recent inbreeding events.
the significant correlation between the sharing of roh regions and regions putatively under selection   suggests that some of these short shared roh are the result of a combination of inbreeding and selection.
instead of roh regions randomly distributed over the genome, there were dense-roh peak regions in the actual sharing of roh regions among individuals , which supports the hypothesis that the observed roh patterns are not solely a result of demography , as random roh distributions would only be expected as a result of inbreeding.
distribution of functional variants in the cattle genome
years of intensive artificial selection in cattle breeding have resulted in reduced genetic diversity in cattle populations as demonstrated by the high proportion of roh  found in this study.
however, the results regarding roh patterns suggest that the distribution and enrichment of putative functional variants in different roh lengths is more interesting.
szpiech et al.
suggested that damaging variants are more enriched in human roh, particularly longer roh  <cit> .
the observed speed of deleterious homozygote accumulation in roh far exceeds the accumulated decrease in deleterious homozygotes in non-roh regions .
this is expected, since identity by decent causes homozygosity to increase in roh regions compared to non-roh regions.
deleterious variants were expected at a low frequency; therefore, rare occurrences of deleterious alleles were expected in the homozygous state.
however, when a stretch of homologous dna fragments are identical by descent, the probability of deleterious alleles increases .
we also observed a higher allele frequency of deleterious variants in roh regions compared to non-deleterious variants .
this was also expected as increased deleterious variants occur when allele frequencies are extreme, as has been observed in humans  <cit> .
in cattle, variants with ‘deleterious’ effects on protein structure may be artificially selected more frequently due to economic benefits.
one example of this is a myostatin gene mutation in belgian blue cattle resulting in a “double muscling” phenotype.
meat from these cattle has a reduced fat content, as the mutation converts feed into increased lean muscle  <cit> .
cattle populations have been under strong artificial selection for many generations.
the significant correlation between sharing of roh regions and regions putatively under selection pressure   confirms that some of the shared short roh regions have been selected and spread throughout the population.
moreover, by randomization of roh regions over the genomes, additional file 17: figure s12b presented the patterns of roh were only result of inbreeding.
however, we do observe several dense-roh peak regions shared among individuals in our populations, further supporting our belief that roh patterns are not only the result of pure demography.
therefore, the distribution pattern and abundance of functional variants in different roh lengths in cattle likely differs from the human population.
artificial selection purges deleterious alleles from regions that frequently occur in roh, favoring alleles with strong beneficial effects.
specifically, the interaction between inbreeding and artificial selection for particular variants can have a strong effect on the distribution of functional variants.
moreover, potentially deleterious mutations might hitchhike with selected variants.
long-term artificial selection enriches cattle populations with beneficial alleles in short and medium roh, along with hitchhiked deleterious variants, while variants in long roh remain neutral.
the proportion of predicted deleterious homozygotes was greater in roh regions than non-deleterious homozygotes.
however, predicted deleterious homozygotes varied by the length of roh.
the rate of change differed between small, medium, and long roh.
higher rates were observed in short and medium compared to long roh.
the slopes for deleterious and non-deleterious homozygotes were significantly different in short and medium roh, and the patterns were similar for predicted nonsense  and loss of function  variants .
we also examined patterns of deleterious homozygote frequency in roh using different length thresholds and saw the similar trends as reported using our original length thresholds .
predicted deleterious variants may be detrimental, however, these allele frequencies cannot increase in long roh, as inbred individuals harboring a large proportion of long roh with a high frequency of deleterious alleles will have reduced fitness, leading to decreased survival.
alternatively, predicted deleterious variants may be harmful alleles that were carried into the genome with artificially selected beneficial alleles, and were therefore favored by selection over a number of generations and are reflected in short or medium roh .
long roh are evidence of recent shared ancestry, while short roh typically reflect more ancient relatedness  <cit> .
long roh regions have gone through selection for few generations, and will eventually break down into medium and then short roh.
allelic combinations will likely be recombined within a few generations and disappear due to segregation  <cit> .
in contrast, deleterious short or medium roh variants, which reportedly hitchhike with beneficial alleles, are thought to persist for an extended periods of time and are shared among individuals via gene flow   <cit> .
some of these shared short roh regions were observed to be overlapping with regions under selection based on the fst analysis and ihs testing .
one mechanism for this is when a beneficial mutation occurs in a population and then spreads to the entire population, forming a selective sweep.
artificial selection will favor short or medium roh regions harboring beneficial mutations that will spread and eventually become fixed in the sampled populations .
therefore, we deduce that some predicted deleterious homozygotes in short or medium roh were deleterious alleles that hitchhiked with beneficial variants and were selected in the population.
alternatively, shorter roh may represent the interplay between random inbreeding and artificial selection for particular variants.
therefore, some of these shared short roh tended to be candidate regions for selection.
however, homozygosity for certain short and medium roh regions were not maintained due to the absence of selection for any specific alleles; therefore, variants with deleterious effects will be purged by artificial selection.
it should also be noted that the confounding effect of inbreeding with selection in generating long stretch of haplotype homozygosity may influence the robustness of ehh-based tests.
lohmueller et al.
<cit>  suggested human populations with decreased genetic diversity supported an excess of recessive deleterious variants, resulting from founder effects in ancient populations during speciation  <cit> , with inflation in the frequency of these rare variants in contemporary populations.
we observed a higher proportion of deleterious than non-deleterious homozygotes in roh .
therefore, another possible explanation for these results is a history of population inbreeding and founder events , with preservation of deleterious variants from ancient populations in contemporary cattle populations represented by our samples.
however, artificial selection has been implemented in cattle populations for many years, and regions under selection pressure tend to overlap with short, shared roh regions .
this suggests that these preserved ancient alleles may have carried deleterious alleles via hitchhiking.
predicting how variation affects gene function has varying degrees of reliability.
sift scores  <cit>  were used to estimate potential fitness consequences for non-reference alleles in our analysis.
certainty regarding predicted functional effects is based on changes in the primary amino acids and impacts on protein function and biological processes.
however, here we examined functional variant distribution in roh regions instead of exploring the effects of each deleterious variant on fitness.
a general pattern was obtained by combining all deleterious or non-deleterious variants into one specific class to explore variant distribution in roh regions.
furthermore, our observations were confirmed by nonsense and loss of function variant classification.
similar patterns were observed when grouping variants into nonsense and loss of function variants.
it should be noted we only emphasized non-reference homozygotes with substantial effects on the organism and did not determine the impacts of reference homozygotes.
there is the potential for deleterious or selected reference homozygotes, and, therefore, these alleles should also be examined.
however, reference alleles are not annotated with a sift score, preventing their examination in this study.
CONCLUSIONS
we characterized roh using genome sequence data in four cattle breeds.
the genome-wide proportion and distribution patterns of roh differed among hol, jer, new-red, and old-red cattle breeds.
we observed a significant correlation between the shared short roh regions and regions putatively under selection.
we also showed the distribution of functional variants in different roh regions and an increased frequency in predicted deleterious homozygotes in short and medium, but not long, roh, which differs from the human genome.
however, the observed pattern and distribution of functional variants is consistent with the population history of the cattle studied, and we suspect that the observed distribution of functional variants is a result of combination of inbreeding and long-term artificial selection in cattle populations.
this is supported by the significant correlation between shared short roh regions and regions putatively under selection.
our findings contribute to the understanding of the effects of inbreeding and probably selection on shaping the distribution of functional variants in the cattle genome.

 dairy cattle have been subjected to more than 60 years of intense selection for traits to enhance milk production. relatively few bulls were chosen to produce thousands of daughters, resulting in large half-sib families. traditionally, cattle breeders estimate the inbreeding coefficient by the degree of parental relatedness using pedigree and genotype data. the rate of inbreeding in cattle populations has increased in recent years, and there is a strong correlation between inbreeding levels and reduced fitness. this can be explained by the increased risk of homozygosity for deleterious alleles as inbreeding increases. thus, high levels of inbreeding in populations will result in inbreeding depression. reduced variability also leads to a reduced selection response in breeding programs. thus, maintaining genetic diversity is crucial in cattle breeding populations. the availability of high-throughput whole genome sequencing for substantial numbers of animals has opened new avenues in examining genetic diversity and led to reliable and detailed investigation of large chromosome segments, including stretches of homozygous genomic regions. runs of homozygosity are contiguous homozygous stretches in an individual genome due to transmission of identical haplotypes from parents to offspring. roh detection can be used to improve mating systems and minimize inbreeding. however, the effects of inbreeding vary among individuals and populations, and it has long been of interest to explore the mechanisms of inbreeding depression and deleterious variants at the genomic level. recently, roh extracted from snp chip data have been used to study the population history of different cattle breeds. purfield et al. claimed that both natural and artificial selection of cattle, as well as demographic processes, have resulted in breeds with extensive phenotype variation. roh may provide useful information on how these processes work in disparate populations, especially for cattle due to recent intense selection of sires, artificial insemination, and embryo transfer in some cattle breeds. however, roh estimated from snp chip data may miss short and medium roh due to limited resolution. additionally, snp arrays suffer from ascertainment biases due to inclusion of snps with high minor allele frequencies. consanguineous mating, population size reduction, and selection result in long homozygous regions along the genome. two copies of a genome segment in an individual inherited from a common ancestor without recombination are identical-by-descent. roh that arise due to inbreeding tend to be fairly long and are dispersed more or less randomly throughout the genome. accumulation of deleterious variants by definition creates fitness consequences, particularly when homozygous. distribution of functional homozygote variants provides information on enrichment of deleterious homozygotes within homozygous regions compared with neutral homozygotes. charlesworth et al. suggested that a large number of weakly deleterious variants are purged by negative selection. a human genome study indicated that purifying selection interacted with founder effects during demographic processes, affecting the proportion of recessive deleterious variants. recently, based on variant annotations, szpiech et al. reported long roh enriched deleterious variation in the human genome. by counting deleterious and neutral variants inside and outside roh in humans, they determined that out of the two competing hypotheses regarding patterns of deleterious variation in the human genome — namely, whether a smaller or larger proportion of all deleterious homozygotes resided in roh regions compared with neutral homozygotes, a larger proportion of deleterious homozygote variants were found, and deleterious variants in long roh accumulated more rapidly compared to short and medium roh in the human genome. cattle domestication was initiated approximately  years ago, with evidence of at least two separate domestication events. during the last few decades, breeders in modern dairy cattle breeding programs have implemented strong artificial selection. kim et al. showed that selection increases overall autozygosity across the genome, whereas the autozygosity in an unselected line does not change significantly across most chromosomes in cattle populations. genomic regions under positive selection show increased roh levels due to local reductions in haplotype diversity, i.e. selective sweeps. certain haplotypes under these conditions constitute a large proportion of the total haplotype pool for the portions of the genome targeted for selective sweeps. in these conditions, a portion of the roh is the actual selection target and is retained in the roh region. these roh will increase in frequency and be subject to purging, as the spread of haplotypes carrying deleterious recessive alleles are restrained by forces counteracting the selection pressure driving the selective sweep. therefore, we expect that this roh type would be on average shorter, composed of more common haplotypes, be concentrated in fewer genomic locations, and contain fewer deleterious alleles than the genomic average. short roh are generally due to older haplotype relatedness, while longer roh result from more recent parental relatedness. thus, short and medium sized roh have been subject to selection for a longer period of time than longer roh. furthermore, recombination will have had more time to trim down roh that have been the target of selective sweeps. roh regions are expected to exhibit an increased frequency of homozygotes compared to non-roh regions, as the homozygote allelic frequency in non-roh regions is p and the allelic frequency in roh regions is p, given a population allelic frequency of p. therefore, ibd results in enrichment of homozygotes in roh. given the nature of cattle breeding, artificial selection is expected to play a more crucial role in shaping the frequency and distribution of functional variants in roh in modern cattle relative to human populations. therefore, we expect that selection pressures could reduce the number of regions with an increased frequency of deleterious variations, and, at the same time, enrich for short roh regions with substantial beneficial effects in cattle breeding populations. this remains an ongoing process, as the properties of genomic variants within roh regions continue to be discovered in cattle. an increase in functional variant frequency in different roh length classes should also be examined under different population-genetic processes, such as the number of generations of inbreeding. genome scale bioinformatics annotations are available from a number of sources, including the variants effect predictor from ensembl, with several available levels of annotation. generally, synonymous polymorphisms are the least subject to selective forces as they have the least effect on the resultant protein. tools such as sift can be used to predict non-synonymous change effects on proteins, and, thus, give an idea of their likely selective pressure. sift classifies non-synonymous changes as either non-deleterious or deleterious, based on the predicted effect on the protein. by comparing these classes, we can study how selection has shaped deleterious variants within the cattle genomic roh. we hypothesize that due to strong artificial selection pressures and demographic processes in cattle, deleterious variants increased in frequency within roh compared with non-deleterious variants and that the deleterious allelic frequency and distribution in roh classes differs markedly from the human genome. testing this hypothesis will contribute to our understanding of inbreeding in cattle and help elucidate how artificial selection and other population level processes affect the distribution of functional variants. to accomplish this, we examined patterns of roh detected using full genome sequencing in four danish cattle breeds and studied the distribution and frequency of deleterious and non-deleterious variations in different length roh regions.  general statistics runs of homozygosity in the autosomes of resequenced individuals were determined from four danish dairy cattle breeds: holstein, jersey, old red danish dairy cattle, and new danish red dairy cattle. the average genomic roh content was  % across the four cattle breeds, with hol, jer, new-red, and old-red having  %,  %,  %, and  %, respectively. the average number of roh per genome was  ± , with an average size of.2 bp, ranging from kbp to kbp. the mean roh size varied significantly between hol, jer, old-red, and new-red with the exception of jer and old-red. the mean number of roh was significantly different between hol, jer, old-red, and new-red cattle with the exception of hol and old-red.fig. roh general statistics. a average genome-wide nucleotide diversity; b average nucleotide diversity outside roh; c average roh size; d average genome-wide roh totals the average genome-wide nucleotide diversity was heterozygous positions per kbp across all individuals, including roh, and heterozygous positions per kbp across all individuals in the genome excluding roh. the minimum and maximum nucleotide diversities were snps/kbp in jer and snps/kbp in new-red, respectively and were significantly different between all cattle breeds except hol and new-red. π-out was significantly different between old-red and hol, jer, and new-red. nucleotide diversity was higher in the vicinity of the major histocompatibility complex on chromosome. genomic patterns of homozygosity roh were separated into three size classes: small, medium, and large. the proportion of roh in each size class was computed in all sequenced individuals. while small roh were frequent throughout the genome, they constituted a small proportion of the entire genome. in contrast, medium roh were much less frequent, they constituted significantly more of the genome than either small or large roh. large roh were at least tenfold less numerous than medium roh, but nevertheless covered a sizable proportion of the total genome length. old-red cattle on average had the largest proportion of their genome in large roh. new-red cattle had fewer genomic roh and a smaller genomic proportion of roh than hol and jer cattle. old-red and jer cattle on average had more roh and increased proportion of genomic homozygosity.fig total roh number and genome proportions. a the average small, medium, and large roh numbers for the four breeds; b average total genome roh coverage for a given size class within each breed principal component analysis successfully differentiated the four cattle breed individuals into different clusters based on ssr sequence data, with jer the most distantly related based on pca results. three-dimensional plots did not show clear separation of cattle breeds based on to roh size, roh number, and π-out. new-red cattle represented the most variable cluster due to high nucleotide diversity and fewer roh. there was lower nucleotide diversity and more total roh in jer compared to hol and new-red. despite its most distant origin, jer had lower nucleotide diversity, creating several clusters in the three dimensional plot. however, all danish cattle breeds were more or less clustered together, with the exception of two new-red individuals. based on sequence data, new-red cattle exhibited the fewest roh and smallest roh sizes. this is a composite breed with contributions from other red breeds, including swedish red, finnish ayrshire, and brown swiss. compared with new-red, these data suggest that the old-red breed has been more inbred based on relatively high coverage of long regions of homozygosity, probably due to a relatively small breeding population and recent years of close mating. furthermore, the sharing of roh regions was examined among sequenced individuals. sharing of roh regions primarily happened in short rather than long roh regions, likely a result of combination of inbreeding and selection. significant correlations were observed between fst, ihs, and shared roh regions in bins of 500 kb compared with the whole genome average. we also observed that instead of randomly distributed over the genomes, there were several obvious roh-dense peaks distributed shared between individuals across genomes. therefore, the distribution of roh is not only result of pure demography, but likely the result of selection. distribution of functional variants in roh regions number of deleterious homozygous genotypes in roh additional file 11: table s and additional file 12: table s show the counts for reference homozygotes, heterozygotes, and non-reference homozygotes at deleterious and non-deleterious sites, respectively, that were contained within roh and non-roh regions. the number of deleterious non-reference homozygotes was consistent with the roh coverage in all four breeds. old-red and jer showed increased roh coverage, with a higher number of non-reference deleterious homozygotes in the genome. non-reference non-deleterious homozygotes also exhibited the same trends as deleterious homozygotes. figure  shows the total number of deleterious non-reference homozygotes as a function of the total proportion of the genome covered by roh for all sequenced individuals. as roh coverage increased, a greater number of homozygotes were observed within roh, which was consistent with findings from the human genome. there was a very strong positive correlation between the number of deleterious homozygotes and the genomic roh proportion. similarly, the number of homozygotes outside of roh decreased with the genomic roh proportion due to smaller non-roh regions as roh coverage increased. as expected, there was a weak negative correlation between deleterious homozygotes outside roh and the genomic roh proportions. compared with data from the human genome, the decreased slope for non-roh regions was much shallower than the increased slope for roh regions. however, this indicates that the increased deleterious homozygotes in roh regions exceed deleterious homozygote declines in non-roh regions in cattle. similar to the human genome, the fitted lines also predict that, on average, individual non-inbred cattle carry approximately deleterious homozygous variants. an increased in roh coverage by 10 % will increase the expected deleterious homozygote numbers in roh regions by and decrease the expected number of deleterious homozygotes in non-roh regions by yielding an expected net increase of deleterious homozygotes.fig. deleterious non-reference homozygotes versus the genome roh coverage in each individual. red points represent the number of deleterious homozygotes falling within roh regions and black points represent the number of deleterious homozygotes falling outside roh regions deleterious and non-deleterious homozygotes in roh of any size figure 4a shows the proportion of deleterious non-reference homozygotes inside and outside roh regions versus total genomic roh coverage. the proportions of non-deleterious and deleterious homozygous genotypes within roh were strongly positively correlated with total genomic roh coverage. these high correlations were expected, because as larger proportions of homozygous genotypes occur, roh coverage in the genome increases, and therefore roh comprise an increasingly greater proportion of the genome. the fi,rd proportion in genome-wide deleterious homozygotes within roh consistently exceeded the fi,rn proportion of genome-wide non-deleterious homozygotes within roh and the increasing slopes differed between deleterious and non-deleterious variants.fig. the proportion of all genome-wide non-reference homozygotes falling in roh regions versus the genome roh coverage for each individual. a any roh region; b short; c medium; and d long roh regions. red points represent deleterious homozygotes, and blue points represent non-deleterious homozygotes after fitting the two linear regression models, we found β was significant indicating that the interaction between the two regression slopes was significant. however, β was not significant between the two regression intercepts. this is consistent with previous findings in humans, where deleterious homozygotes showed increased frequency in roh relative to non-deleterious homozygotes and regression slopes were significantly different between deleterious and non-deleterious homozygotes. deleterious and non-deleterious homozygotes by roh size figure 4b shows fi,sd and fi,sn versus total genomic coverage for small roh . the proportion of non-deleterious homozygous genotypes in small roh, and the proportion of deleterious homozygous genotypes in small roh were positively correlated with genomic coverage. figure 4c and d showed fi,md and fi,mn versus total genomic coverage for medium and large roh. the regressions for homozygote numbers in medium and large roh had smaller p-values than in small roh. these results show that deleterious homozygotes occur more frequently in roh than non-deleterious homozygotes. additionally, when the proportion of deleterious homozygotes within large roh is compared to the proportion within small roh, there was a substantially higher proportion of genome-wide deleterious homozygotes in small and medium vs. large roh especially in individuals with moderate to high roh coverage proportions. given that roh coverage for all individuals across the four breeds differed. therefore, statistical tests for each size group were robust across the breeds and there were different roh coverage groups across all individuals. similar trends were observed for each roh size group, and significantly different degrees of enrichment were observed within each size group.fig. the genome-wide proportion of all non-reference homozygotes falling in different roh sizes versus genome roh coverage for each individual. red, orange, and black points represent deleterious homozygotes in large, medium, and small roh regions, respectively the intercepts and slopes of deleterious homozygotes and non-deleterious homozygotes were significantly different for large and medium roh, but slopes and intercepts were not significantly different between small and medium roh. these results indicate inbreeding that generates short and medium roh increases the proportions of deleterious and non-deleterious homozygotes in roh regions compared to long roh. if deleterious, non-deleterious, and synonymous homozygotes are considered as three separate classes, patterns similar to those observed in the deleterious and non-deleterious homozygotes analysis emerge. deleterious homozygotes were at highest proportions in short and medium length roh. there were smaller proportions of synonymous and non-deleterious homozygotes as genomic roh coverage of small and medium roh increases. in large roh, the synonymous homozygote proportion over all homozygotes was higher than deleterious and non-deleterious homozygote proportions. nonsense variants and roh additional file 13: table s and additional file 14: table s report nonsense and loss of function nonsense sites, respectively, with counts for reference homozygotes, heterozygotes, and non-reference homozygotes, which fell into roh and non-roh regions. figure 6a shows nonsense mutation distribution across all roh. for low-roh individuals, the mean proportion of non-deleterious homozygote variants falling in roh marginally exceeded the nonsense or loss of function variants. for high-roh individuals, however, the proportion of non-deleterious homozygotes within roh was lower than for nonsense homozygotes. when roh are segregated by size, including individuals with high genomic roh coverage, the proportion of nonsense homozygotes in medium roh was greater than that of non-deleterious homozygotes, while the proportion of nonsense homozygotes was slightly lower than non-deleterious homozygotes for large roh. this is consistent with the finding that high-roh individuals exhibited an increased proportion of damaging homozygotes in roh of any size, primarily driven by medium roh.fig. the proportion of all genome-wide non-reference homozygotes falling in roh regions for non-deleterious variants, nonsense variants, and loss of function nonsense variants versus the genome roh coverage for individuals in the “low roh” and “high roh” groups. a: any roh region; b: short; c: medium; and d: long roh regions discussion cattle genomic roh patterns the individuals re-sequenced in the present study are key ancestors from the four danish cattle breeds. the new-red is a composite breed with its primary origins in the old danish red, and includes contributions from other red breeds, including swedish red finnish ayrshire, and brown swiss cattle. our results showed roh size ranged from tens of kb to several mb and varied among individuals from different cattle breeds. overall, medium sized roh were most common. the average proportion of the genome represented by roh was  % across all sequenced individuals. however, pedigree records and lower density snp chips underestimated the inbreeding coefficient for these bulls compared with that generated using roh from genome sequencing. a previous study in danish cattle also reported a less than 5 % inbreeding coefficient using pedigree information. among breeds, jer and old-red were relatively more inbred compared with hol based on high roh genome coverage. meanwhile, a high number and proportion of long roh were detected in old-red, likely indicating a small population with close mating for some period of time. however, the smallest proportion of roh was detected in new-red, presumably due to its outbred origins. these roh patterns are consistent with the known population history for these four breeds. pemberton et al. allocated humans from different demographics to their corresponding place of origin by inferring population history from roh analysis. in our study, we also determined that individuals with similar roh distributions clustered together due to similar patterns of variation. in contrast, pca generated different cluster types in the same sets of individuals based on population structure. the fact that nucleotide diversity outside roh was higher in old-red than hol, jer, and new-red indicates that the historic genetic diversity of old-red might be relatively higher than the other breeds examined due to larger breeding populations for each of several past generations. furthermore, evidence suggested old-red was the source population of new-red. although the newly derived red populations exhibited the highest nucleotide diversity across the entire genome, the ancient haplotypes were more diverse in the ancestral old-red population. reduced nucleotide diversity outside roh in new-red may also be explained by gene flow from hol to new-red. in addition, nucleotide diversity levels outside roh presumably reflected the different origin of old-red from hol and jer. bovine major histocompatibility complex regions have long been known in the cattle genome. our analysis also detected mhc regions, which have a high degree of nucleotide diversity, on chromosome. pemberton et al. reported a correlation between linkage disequilibrium levels and roh distribution in the human genome. mhc regions contain recombination hotspots and, therefore, high recombination rates to maintain relatively high levels of genetic diversity, but are subject to over dominant selection. roh are rarely present in mhc regions of the cattle genome, preventing random distribution of roh. purfield et al. showed roh patterns in cattle populations using snp chip data. our analysis is the first to use next-generation sequencing data to infer roh in cattle and indicate that long roh can only be detected with 50 k or hd snp chip data, while short roh are not detectable due to the required density of snp chip data. short roh regions shared between individuals in our data confirm that short roh were selected and derived from ancient haplotypes that became fixed in populations, while long roh are the result of more recent inbreeding events. consequently, snp chip data misses information more relevant to historic inbreeding practices rather than recent inbreeding events. the significant correlation between the sharing of roh regions and regions putatively under selection suggests that some of these short shared roh are the result of a combination of inbreeding and selection. instead of roh regions randomly distributed over the genome, there were dense-roh peak regions in the actual sharing of roh regions among individuals, which supports the hypothesis that the observed roh patterns are not solely a result of demography, as random roh distributions would only be expected as a result of inbreeding. distribution of functional variants in the cattle genome years of intensive artificial selection in cattle breeding have resulted in reduced genetic diversity in cattle populations as demonstrated by the high proportion of roh found in this study. however, the results regarding roh patterns suggest that the distribution and enrichment of putative functional variants in different roh lengths is more interesting. szpiech et al. suggested that damaging variants are more enriched in human roh, particularly longer roh. the observed speed of deleterious homozygote accumulation in roh far exceeds the accumulated decrease in deleterious homozygotes in non-roh regions. this is expected, since identity by decent causes homozygosity to increase in roh regions compared to non-roh regions. deleterious variants were expected at a low frequency; therefore, rare occurrences of deleterious alleles were expected in the homozygous state. however, when a stretch of homologous dna fragments are identical by descent, the probability of deleterious alleles increases. we also observed a higher allele frequency of deleterious variants in roh regions compared to non-deleterious variants. this was also expected as increased deleterious variants occur when allele frequencies are extreme, as has been observed in humans. in cattle, variants with ‘deleterious’ effects on protein structure may be artificially selected more frequently due to economic benefits. one example of this is a myostatin gene mutation in belgian blue cattle resulting in a “double muscling” phenotype. meat from these cattle has a reduced fat content, as the mutation converts feed into increased lean muscle. cattle populations have been under strong artificial selection for many generations. the significant correlation between sharing of roh regions and regions putatively under selection pressure confirms that some of the shared short roh regions have been selected and spread throughout the population. moreover, by randomization of roh regions over the genomes, additional file 17: figure s12b presented the patterns of roh were only result of inbreeding. however, we do observe several dense-roh peak regions shared among individuals in our populations, further supporting our belief that roh patterns are not only the result of pure demography. therefore, the distribution pattern and abundance of functional variants in different roh lengths in cattle likely differs from the human population. artificial selection purges deleterious alleles from regions that frequently occur in roh, favoring alleles with strong beneficial effects. specifically, the interaction between inbreeding and artificial selection for particular variants can have a strong effect on the distribution of functional variants. moreover, potentially deleterious mutations might hitchhike with selected variants. long-term artificial selection enriches cattle populations with beneficial alleles in short and medium roh, along with hitchhiked deleterious variants, while variants in long roh remain neutral. the proportion of predicted deleterious homozygotes was greater in roh regions than non-deleterious homozygotes. however, predicted deleterious homozygotes varied by the length of roh. the rate of change differed between small, medium, and long roh. higher rates were observed in short and medium compared to long roh. the slopes for deleterious and non-deleterious homozygotes were significantly different in short and medium roh, and the patterns were similar for predicted nonsense and loss of function variants. we also examined patterns of deleterious homozygote frequency in roh using different length thresholds and saw the similar trends as reported using our original length thresholds. predicted deleterious variants may be detrimental, however, these allele frequencies cannot increase in long roh, as inbred individuals harboring a large proportion of long roh with a high frequency of deleterious alleles will have reduced fitness, leading to decreased survival. alternatively, predicted deleterious variants may be harmful alleles that were carried into the genome with artificially selected beneficial alleles, and were therefore favored by selection over a number of generations and are reflected in short or medium roh. long roh are evidence of recent shared ancestry, while short roh typically reflect more ancient relatedness. long roh regions have gone through selection for few generations, and will eventually break down into medium and then short roh. allelic combinations will likely be recombined within a few generations and disappear due to segregation. in contrast, deleterious short or medium roh variants, which reportedly hitchhike with beneficial alleles, are thought to persist for an extended periods of time and are shared among individuals via gene flow. some of these shared short roh regions were observed to be overlapping with regions under selection based on the fst analysis and ihs testing. one mechanism for this is when a beneficial mutation occurs in a population and then spreads to the entire population, forming a selective sweep. artificial selection will favor short or medium roh regions harboring beneficial mutations that will spread and eventually become fixed in the sampled populations. therefore, we deduce that some predicted deleterious homozygotes in short or medium roh were deleterious alleles that hitchhiked with beneficial variants and were selected in the population. alternatively, shorter roh may represent the interplay between random inbreeding and artificial selection for particular variants. therefore, some of these shared short roh tended to be candidate regions for selection. however, homozygosity for certain short and medium roh regions were not maintained due to the absence of selection for any specific alleles; therefore, variants with deleterious effects will be purged by artificial selection. it should also be noted that the confounding effect of inbreeding with selection in generating long stretch of haplotype homozygosity may influence the robustness of ehh-based tests. lohmueller et al. suggested human populations with decreased genetic diversity supported an excess of recessive deleterious variants, resulting from founder effects in ancient populations during speciation, with inflation in the frequency of these rare variants in contemporary populations. we observed a higher proportion of deleterious than non-deleterious homozygotes in roh. therefore, another possible explanation for these results is a history of population inbreeding and founder events, with preservation of deleterious variants from ancient populations in contemporary cattle populations represented by our samples. however, artificial selection has been implemented in cattle populations for many years, and regions under selection pressure tend to overlap with short, shared roh regions. this suggests that these preserved ancient alleles may have carried deleterious alleles via hitchhiking. predicting how variation affects gene function has varying degrees of reliability. sift scores were used to estimate potential fitness consequences for non-reference alleles in our analysis. certainty regarding predicted functional effects is based on changes in the primary amino acids and impacts on protein function and biological processes. however, here we examined functional variant distribution in roh regions instead of exploring the effects of each deleterious variant on fitness. a general pattern was obtained by combining all deleterious or non-deleterious variants into one specific class to explore variant distribution in roh regions. furthermore, our observations were confirmed by nonsense and loss of function variant classification. similar patterns were observed when grouping variants into nonsense and loss of function variants. it should be noted we only emphasized non-reference homozygotes with substantial effects on the organism and did not determine the impacts of reference homozygotes. there is the potential for deleterious or selected reference homozygotes, and, therefore, these alleles should also be examined. however, reference alleles are not annotated with a sift score, preventing their examination in this study.  we characterized roh using genome sequence data in four cattle breeds. the genome-wide proportion and distribution patterns of roh differed among hol, jer, new-red, and old-red cattle breeds. we observed a significant correlation between the shared short roh regions and regions putatively under selection. we also showed the distribution of functional variants in different roh regions and an increased frequency in predicted deleterious homozygotes in short and medium, but not long, roh, which differs from the human genome. however, the observed pattern and distribution of functional variants is consistent with the population history of the cattle studied, and we suspect that the observed distribution of functional variants is a result of combination of inbreeding and long-term artificial selection in cattle populations. this is supported by the significant correlation between shared short roh regions and regions putatively under selection. our findings contribute to the understanding of the effects of inbreeding and probably selection on shaping the distribution of functional variants in the cattle genome. 
by investigating the relationship between roh and the predicted deleterious and non-deleterious variants, we gained insight into the distribution of functional variation in inbred regions. predicted deleterious variants were more enriched in roh regions than predicted non-deleterious variants, which is consistent with observations in the human genome. there were an average of roh per genome with an average size of ~ kbp, ranging from to kbp. recent developments in sequencing technology have facilitated widespread investigations of genomic variants, including continuous stretches of homozygous genomic regions. there was a significant correlation between shared short roh regions and regions putatively under selection. these patterns are different from those in the human genome but consistent with the natural history of cattle populations, which is confirmed by the significant correlation between shared short roh regions and regions putatively under selection. in the present study, roh were characterized in four cattle breeds with whole genome sequence data and the distribution of predicted functional variants was detected in roh regions and across different roh length classes. on average,  % of the genome was located in roh across four cattle breeds. we also found that increased enrichment of deleterious variants was significantly higher in short and medium roh regions compared with long roh regions, which is different than what has been observed in the human genome. this study illustrates the distribution of roh and functional variants within roh in cattle populations. these findings contribute to understanding the effects of inbreeding and probably selection in shaping the distribution of functional variants in the cattle genome. for cattle, a large proportion of these runs of homozygosity are likely the result of inbreeding due to the accumulation of elite alleles from long-term selective breeding programs.
 recent developments in sequencing technology have facilitated widespread investigations of genomic variants, including continuous stretches of homozygous genomic regions. for cattle, a large proportion of these runs of homozygosity are likely the result of inbreeding due to the accumulation of elite alleles from long-term selective breeding programs. in the present study, roh were characterized in four cattle breeds with whole genome sequence data and the distribution of predicted functional variants was detected in roh regions and across different roh length classes.  on average,  % of the genome was located in roh across four cattle breeds. there were an average of roh per genome with an average size of ~ kbp, ranging from to kbp. there was a significant correlation between shared short roh regions and regions putatively under selection. by investigating the relationship between roh and the predicted deleterious and non-deleterious variants, we gained insight into the distribution of functional variation in inbred regions. predicted deleterious variants were more enriched in roh regions than predicted non-deleterious variants, which is consistent with observations in the human genome. we also found that increased enrichment of deleterious variants was significantly higher in short and medium roh regions compared with long roh regions, which is different than what has been observed in the human genome.  this study illustrates the distribution of roh and functional variants within roh in cattle populations. these patterns are different from those in the human genome but consistent with the natural history of cattle populations, which is confirmed by the significant correlation between shared short roh regions and regions putatively under selection. these findings contribute to understanding the effects of inbreeding and probably selection in shaping the distribution of functional variants in the cattle genome. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords runs of homozygositypolymorphismsinbreedingcattlegenome sequencingissue-copyright-statement© the author 2015 