BACKGROUND
sheep , primarily raised for meat, wool, milk, and pelts, are an important part of the agricultural economy worldwide.
it is the first grazing animal known to have been domesticated  <cit> .
archaeological evidence suggests that sheep were probably first domesticated in the fertile crescent, approximately  <dig>  years ago  <cit> .
china is one of the nine independent food producers worldwide  <cit> , and has a long history of sheep husbandry.
the earliest record of chinese sheep remains can be traced back to approximately 5000– <dig> years ago  <cit> .
china has a great diversity of ecosystems and an abundance of sheep resources.
based on geographical distribution and morphological characteristics, there are  <dig> indigenous sheep breeds in china, which can be classified into three groups: mongolian, kazakh, and tibetan  <cit> .
the native domestic sheep breeds are highly adapted to local environmental conditions, and their most distinctive feature is the type of tail.
mongolian  sheep are abundant in high latitudes; kazakh  sheep have the ability to deposit a large amount of fat in the body to meet nutritional demands during the winter and spring; the tibetan  sheep are generally present at low latitudes  where it is warmer and experiences less snow.
in addition, artificial selection is also an important driving force for the formation of species diversity.
most of the chinese domestic sheep are reared for meat, while some varieties are multipurpose.
for instance, duolang is an excellent mutton producer, tibetan sheep are one of the major breeds for carpet wool in china, and hu and large-tailed han are prolific lambskin-type breeds.
in a previous microsatellite analysis, ma et al.
<cit>  indicated that chinese northern sheep could be divided into two broadly defined lineages, tibetan origin and mongolia origin, representing different geographical clusters.
zhong et al.
<cit>  revealed three major clusters of chinese indigenous sheep  and pointed out that chinese indigenous sheep have a complicated genetic structure under the effects of different breeding histories, geographical distributions and ecological factors.
in the present study, we investigated  <dig> chinese indigenous breeds that represent the main sheep types in china using the illumina ovine snp <dig> genotyping beadchip.
our goal was to analyse the population structure and genome selection among chinese native sheep breeds.
RESULTS
genetic variation and population genetic analysis
in this study, four metrics were used to estimate levels of within-breed genetic diversity .
the polymorphism , expected heterozygosity , observed heterozygosity , and inbreeding coefficients  among ten sheep populations were  <dig> – <dig> ,  <dig> – <dig> ,  <dig> – <dig> , and  <dig> – <dig> , respectively.
levels of polymorphism were generally high, more than 92% of loci displaying polymorphism within each population.
the values of expected heterozygosity were close to observed heterozygosity in all populations, and the duolang population had the lowest values for heterozygosity.
moreover, the he of plateau-type tibetan and valley-type tibetan presented were consistent with previous reports  <cit> .
the lowest inbreeding coefficients were observed in kazakh sheep .
we then estimated the ranges of minor allele frequency  for all sheep breeds .
in all breeds, about 40% of single nucleotide polymorphisms  are highly variable  and 15% exhibit limited variability .
finally, we investigated the extent of linkage disequilibrium , as estimated by the average distance between snps that correspond to different linkage disequilibrium r <dig>  in each breed .
the range of average distance was estimated as  <dig> – <dig>  kb and decreased with the increasing of r <dig> value.
and the standard deviation was highest when r2 =  <dig> .
to further examine the relationships among individuals, we performed principal components analysis  among all individuals .
the analysis showed two principal components , with variances of  <dig> % and  <dig> % , respectively.
according to pc <dig>  chinese sheep could be divided into two groups consistent with their fat deposition: thin-type sheep  and fat-type sheep .
they are further subdivided into two different sub-groups distinguished by differences in fat deposition.
second, combining the two principal components  clustered the three tibetan sheep  together and the five mongolian sheep  together; however, the two kazakh sheep  were clearly segregated .
we could distinguish the three breeds in the tibetan group, but it was more difficult in the mongolian group, which was mixed together .figure  <dig>
animals clustered on the basis of principal component  analysis using individual genotypes.
plot for the first  and second  component revealed the clustering of  <dig> animals from uji, hus, ton, lth, lop, kaz, dul, diq, tibp, and tibv breeds; uji: ujimqin sheep, hus: hu sheep, ton: tong sheep, lth: large-tailed han sheep, lop: lop sheep, kaz: kazakh sheep, dul: duolang sheep, diq: diqing sheep, tibp: plateau-type tibetan sheep, and tibv: valley-type tibetan sheep.
all pairwise fst values, which were rescaled as fst / , were calculated between the ten populations .
the lowest level of differentiation was found between the plateau-type tibetan and valley-type tibetan populations , while the greatest divergence was observed between diqing and duolang .
in the mongolian sheep populations, fst between lop and ujimqin was the lowest .
in comparison, the pairwise fst within the groups showed a closer relationship than between the groups.
we calculated the mean pairwise fst  of each breed, which indicated that ujimqin had the lowest mpf  and duolang had the largest mpf .
the nj-tree showed clearly defined clusters .
diqing, plateau-type tibetan, and valley-type tibetan breeds were found in one main branch.
this result was well supported by the traditional classification  and their close distribution in the neighboring areas of tibetan and qinghai provinces.
the other main lineage of the nj tree included the mongolian and kazakh group.
in addition, three breeds  from xinxiang clustered in the same branch.
we also constructed a neighbor-joining  tree among the  <dig> individuals .
the results were similar to the nj-tree of the populations and clearly showed that there were no conflicts concerning the origins of individuals assigned to each breed.figure  <dig>
neighbor-joining  phylogeny for  <dig> chinese indigenous sheep breeds based on pairwise
f
st
.
uji: ujimqin sheep, hus: hu sheep, ton: tong sheep, lth: large-tailed han sheep, lop: lop sheep, kaz: kazakh sheep, dul: duolang sheep, diq: diqing sheep, tibp: plateau-type tibetan sheep, and tibv: valley-type tibetan sheep.
to confirm our observation of the degree of divergence, the program structure was applied to estimate the proportion of common ancestry among the  <dig> breeds.
a model-based unsupervised hierarchical clustering of the individuals was analyzed by considering different k numbers  of predefined clusters based on  <dig>  autosomal snps.
the results of bayesian clustering for k =  <dig> indicated that there was a clear transition from the northwest and north china populations  to the southwest and south china populations .
we found that fat-type sheep were green dominated, wherein duolang and kazakh accounted for more than 90%.
the thin-type sheep were red dominated, and diqing accounted for 90% .
this is also consistent with the pca and nj-tree.
furthermore, when the k value became large, some breeds were independent .
at k =  <dig>  duolang tended to be separated from the fat-type group.
in the pairwise fst analysis, the mean pairwise fst of duolang was the highest among the ten chinese indigenous sheep breeds .
thus, duolang might be a population subdivision within the kazakh group.
at k =  <dig>  the kazakh breed separated from the fat-type group.
soon after, large-tailed han, hu, and tong separated one after another from the fat-type group when k = 8– <dig> figure  <dig>
population structure of  <dig> sheep inferred by model-based clustering using structure.
results from k =  <dig> are shown.
signatures of selection in the ovine genome
the di statistic is a new population-genomics strategy based on levels of population differentiation, which determines robustly whether selection acts on newly arisen or pre-existing variations  <cit> .
we performed di statistic analyses to identify candidate regions targeted by selection in  <dig> chinese indigenous sheep breeds.
the di statistic was calculated for autosomal snps in 300-kb windows, with a minimum of three snps per window, and defining the populations by breed.
the di statistic is a summation at each window of pairwise fst values for each breed combination, corrected by the value expected from genome-wide calculations; therefore, a large value of di statistic indicates greater divergence at that 300-kb window than that observed across the genome as a whole.
in total,  <dig>  snps were evaluated within  <dig> windows, averaging  <dig>  snps per window .
we define candidate selection regions that fell into the upper 99th percentile of the empirical distribution.
the  <dig> windows within each breed were considered putative signatures of selection.
in total,  <dig> of the windows met this criterion in one or more of the  <dig> breeds.
these regions, considered putative signatures of selection in each breed, are listed in additional file 8: table s <dig>  the maximum di statistic value per breed ranged from  <dig>  in the kazakh to  <dig>  in the valley-type tibetan.
additional file 9: figure s <dig> shows the genome-wide distribution of the di statistic.
to investigate how many selective events were unique or shared among breeds, we calculated the number of overlapping signatures of selection for each of the  <dig> significant 300-kb windows .
sixteen of the  <dig> significant windows  exhibited signatures of selection in four or more breeds.
sliding-window analyses of pairwise fst across the 300-kb interval suggested two or more independent selective events, reflected by two peaks of differentiation, with distinct patterns of allele frequency divergence among breeds .figure  <dig>
top  <dig> strongly selection regions in chinese indigenous sheep.
the number of overlapping signatures of selection in each 300-bp window.
we defined an overlapping signature of selection for each window if the empirical p value was ≤  <dig>  in one breed.
the black arrow indicates the chromosomal region shown in b and c;  the per-snp d
i statistic of three regions with three consecutive windows, with t <dig>  t <dig> and t <dig> widows in the middle.
the braces indicate the group to which the breed belongs;  the rsb value of each snp in the three regions, which are consistent with b. the black arrow indicates the group name.
the dashed red line denotes the significance threshold at p =  <dig> .
the first peak of differentiated region  of the sheep genome showed evidence of selection in multiple breeds on oar <dig> .
the differentiation observed from  <dig>  to  <dig>  mb coincides with the rxfp <dig> gene as a candidate gene for sheep horns  <cit> .
in the  <dig> window, we found that the 2nd to 4th snp clearly divided  <dig> sheep breeds into two groups , one is horned and the other is mixed and polled.
the horned breeds  showed high levels of differentiation at rxfp <dig> compared with the mixed and polled breeds .
the second peak of differentiation region  in oar <dig> from  <dig>  to  <dig>  mb included the ppp1cc gene, also known as ppp1g, which is a subunit of the protein phosphatase  <dig>  it is a glycogen-associated phosphatase responsible for dephosphorylation and subsequent inactivation of glycogen synthase and is universal in skeletal muscle  <cit> .
ppp1cc is not required for insulin-stimulated glycogen synthesis in skeletal muscle, but appears to be a component of the response to contractile action  <cit> .
recent research indicates that ppp1cc is a positional candidate locus for skeletal muscle strength phenotypes  <cit> .
in the  <dig> di statistic window, three snps showed higher di statistic values in the thin-type breeds  than in the fat-type breeds  .
we analyzed the remaining  <dig> significant regions, which showed selection between four and seven breeds, and found the t <dig> region  in oar <dig>  which was selected in seven breeds, and divided the  <dig> breeds into two groups, identical to the t <dig> region.
this region, from  <dig>  to  <dig>  mb, included only one functional gene: platelet-derived growth factor d , which is one of the four subfamilies of pdgf, and is a potent stimulator of proliferation.
pdgf was first identified  <cit>  and purified  <cit>  from human platelets, where it is sequestered in the alpha granules and released upon platelet activation.
studies have shown that the pdgf gene promotes proliferation and inhibits differentiation of preadipocytes  <cit> .
real-time quantitative pcr indicate that pdgfd is expressed at a higher level in adipose tissue e than in human normal tissues, except the thyroid  <cit> .
to better understand the selection of these three regions, they were divided into two groups based on the di statistic value.
rsb  is a new approach to detect recent positive selection based on contrasting the extended haplotype homozygosity  profiles between populations  <cit> .
we calculated the rsb value based on the groups that were included in each window .
the rsb results showed region t <dig> was selected in horned groups and region t <dig> was subject to selection in fat-type populations; however, neither reached statistical significance.
interestingly, we found some snps before t <dig> and t <dig> windows were under significantly selection in rsb analysis.
the density of ovine 50 k snp beadchip is not high enough and may lead to low detection ability and deviation.
by contrast, region t <dig> was significantly positively selected in the fat-type groups  and the pdgfd gene promotes proliferation.
therefore, we hypothesized that this region may be related to the formation of fat-type sheep.
in addition, we also found an interesting peak of differentiation window in oar <dig> of six selection breeds , which overlaps with two genes, msrb <dig> and lemd <dig>  a snp  in this window, within msrb <dig>  differentiates duolang strongly from the others .
msrb <dig> was identified as a candidate gene for ear morphology in dogs  <cit>  and pigs  <cit>  by genome-wide association studies .
at this locus, duolang and diqing show the two extremes of the allele frequencies .
from the pictures of each breed, we observed that the ears of the duolang sheep are the largest of all the sheep breeds .
figure 5c shows photographs of the two extreme breeds.
therefore, we hypothesized that that this snp is associated with ear size in sheep.table  <dig>
annotation of the top six signatures of selection windows
frequency
breed
region
annotated gene
eef1a <dig> rxfp2
muted,ppp1cc
pdgfd
msrb <dig> lemd3
nf <dig> omg, evi2b
aasdhppt,ankrd <dig> gpr <dig> lrrc <dig> mre11a
breed abbreviations are described in table  <dig>
breeds included in the study and their distinguishing phenotypes
breed
location
group
tail type
horn type
coat color
breeding objective
lambing percentage
all information comes from animal genetic resources in china - sheep and goats.
a snp associated with sheep’s ear morphology.
the per-snp d
i statistic in  <dig> – <dig>  mb on oar  <dig>  the black arrow indicates a snp that is associated with ear size;  the allele frequencies of  <dig> chinese indigenous sheep breeds; red squares represents a and green squares represent g;  duolang and diqing: two extremes of the allele frequencies.
cross traces in duolang
duolang is a special breed that showed a large genetic distance from other chinese indigenous breed in a previous population genetic analysis.
historically, a religious person brought jill wagner sheep from afghanistan to xinjiang in  <dig>  and then crossed them with native sheep breeds, eventually forming a new breed called duolang  <cit> .
the major characteristics are large body size, fast growth and a high reproductive rate  <cit> .
in the present study, we found two consecutive selection regions on oar <dig> and  <dig>  involving a total of  <dig> windows , which were almost half of the candidate selection regions of duolang.
these windows contained  <dig> candidate genes .
in oar <dig> , the highest di statistic window  did not include any genes, and the second highest di statistic window  included nine genes .
npr <dig>  which was found by both kijas and moradi  <cit> , is involved in skeletal morphology and body size  <cit> .
spag <dig> and hint <dig> influence carcass weight and birth body weight, respectively  <cit> .
in oar <dig> , we found that the highest di statistic window  was for a region associated with a milk production qtl in cattle  <cit> , which included four genes , mepe and ibsp).
in particular, spp <dig> is a candidate gene for the litter size in pig  <cit> .figure  <dig>
plot of
d
i
windows of two regions on oar <dig>  and oar <dig>  of  <dig> chinese indigenous sheep breeds.
a black arrow indicates the peak windows in each region.
the braces include the candidate genes.
other important candidate genes selected in less than three breeds
such universal differentiation at a single locus is consistent with the action of a gene that generally sorts individuals into phenotypic classes and breed groups.
in this study, we also found some common selection genes that influence reproduction and coat color.
bmpr1b, located on oar <dig> , showed strong evidence of selection in highly prolific breeds hu and large-tail han.
although bmpr1b is associated with high prolificacy in some chinese breeds or strains of sheep, other researchers believe that bmpr1b is not the only gene responsible for the fecundity of chinese sheep  <cit> .
we also identified that kit, mc1r, and fry influence coat color.
a region on oar <dig>  that includes the kit gene is associated with melanocyte development and accounts for pigmentation phenotypes in pigs and horses  <cit> , which also shows a strong signature of selection in the duolang and kazakh breeds, both of which have dark coat colors .
mc1r was selected in the ujimqin population.
a mutation in mc1r causes black spotting in pigs  <cit> .
fry, which is a key candidate gene involved in the piebald phenotype in merino sheep  <cit> , is located on chromosome  <dig> and was selected in valley-type tibetan sheep, whose coat has black spots .
interestingly, fry was also considered as a candidate gene affecting wool quality between rambouillet and suffolk sheep  <cit> .
furthermore, wnt <dig> and wnt10a, which were only selected in the rumped tail breeds kazakh and duolang, inhibit adipogenesis via a β-catenin-dependent mechanism  <cit> .
discussion
in the present study, population genetic analysis was performed on 50 k snp genotypes of  <dig> animals for  <dig> chinese indigenous sheep breeds.
we analyzed the population using pca, structure, and nj-tree.
the results indicated that chinese indigenous sheep populations could be subdivided into two genetic clusters: the tibetan group and the mongolian and kazakh group.
overall, the partitioning of genetic diversities of the breeds is consistent with their geographic distributions.
the mongolian group is the most widely distributed breed in china, mainly in inner mongolia, the central plains, and eastern coastal areas.
this is attributed to good adaptability and performance of the mongolian sheep and genghis khan’s expedition in the yuan dynasty  <cit> .
the most abundant species diversity is in xinjiang province, which is mainly attributed to its location.
it is the only way to the trading venues in the “silk road”  <cit> , and its position in central asia and the central plains, and the geographic isolation of its southern and northern parts is one reason for its rich diversity.
the tibetan group living in the southwest region, which is a mountainous region, also has abundant genetic diversity.
according to pairwise fst, the relationships between lop and ujimqin  and between plateau-type tibetan and valley-type tibetan  were closer than others.
these two combinations were observed to be mixed together in the pca plot , and had a similar composition in structure from k = 2– <dig> .
the greatest divergence was observed between diqing and duolang , which come from different groups: diqing belongs to the tibetan group and duolang belongs to the kazakh group.
a previous study indicated that the mongolian group was the origin of tong sheep  <cit> .
however, according to our study, ujimqin is the oldest breed in the mongolian population compared with tong sheep, because ujimqin not only has the smallest mpf and the highest polymorphism in the mongolian group, but also it lives closer to mongolia, the capital of the yuan dynasty  <cit> , geographically.
population genetic analysis was able to distinguish physiological differences and geographical origins.
in this study, duolang and kazakh come from xinjiang province; however, their genetic difference was in the middle of the range of differentiation , and similar results were seen from pca and structure .
one reason is that the tianshan mountains divide xinjiang into southern and northern regions: duolang comes from the southern region and kazakh comes from the northern region.
this results in the geographical isolation between duolang and kazakh.
there is another explanation, according to record, duolang was mixed with afghanistan sheep  <cit> , which may be the cause of this phenomenon.
interestingly, we found lop, duolang, and kazakh clustered in a branch of the nj-tree.
however, lop belongs to the mongolian group, according to the pca figures it is closer to mongolian sheep breeds than duolang and kazakh.
in structure result when k = 4– <dig>  lop breed appears to be some introgressed of kazakh group, but this needs more intensive research to explain.
these data provided strong evidence of the genetic structure in these  <dig> sheep breeds.
in this study, which involved just a quarter of chinese local breeds, there were very complex genetic relationships.
for more accurate understanding of the relationships between them, more breeds should be analyzed.
selection in chinese sheep breeds
most studies of artificial selection in sheep have focused on single-gene analyses arising from phenotype-driven studies.
recently, kijas et al.
<cit>  analyzed  <dig> sheep breeds worldwide, one of which was a chinese native breed, tibetan sheep.
the study identified the strongest selected candidate gene, rxfp <dig>  in response to breeding for the absence of horns  <cit> .
we also identified this gene in the peak of the differentiated region in the chinese population.
most chinese indigenous sheep are dual-purpose, low breeding breeds.
only a few specialized breeds are used for lambskin and lamb fur.
hu, large-tailed han and tong are lambskin breed.
hu and large-tailed han are amongst the most prolific breeds in the world.
bmpr1b, a notable candidate gene, was selected in both breeds.
other candidate genes focused on profile characteristics, such as kit, mc1r, and fry, which influence variation in dark coat color and the piebald phenotype.
the fat-tail is an important component in sheep.
currently, approximately 25% of the world sheep population comprises fat-tail breeds, which are found in a wide range of countries in asia, the middle east and north africa  <cit> .
in china, fat-tail breeds comprise 80% of the population.
in terms of tail type, chinese breeds are divided into four groups: short fat-tailed, long fat-tailed, fat-rumped, and thin-tailed .
the first three types of tail shape are not the same, but still belong to fat-type sheep.
this trait is now commercially less important because of improved forage availability and decreased price of the product.
therefore, a decrease in fat-tail size is often desirable for chinese producers.
to date, several investigations into the inheritance of fat-tails have been undertaken.
moradi et al.
<cit>  confirmed three regions located on chromosomes  <dig>   <dig>  and x that affect thin and fat tail breeds.
bakhtiarizadeh et al.
<cit>  suggested that the expression of fabp <dig> in the fat-tail is an important index of fat deposition.
the haplotype in cast can distinguish between fat-tailed and thin-tailed sheep breeds  <cit> .
recently, wang et al.
<cit>  identified  <dig> genes that were differentially expressed between fat-tailed and thin-tailed groups using rna-seq, and identified nell <dig> and fmo <dig> as genes relevant to fat metabolism in adipose tissues.
despite these studies, there is no compelling reason for the impact of a fat-tail on the formation mechanism.
it is worth mentioning that the results of moradi’s  <cit>  and our are inconsistent, even if both of us used similar type of population and the ovine 50 k snp bead chip.
there include two reasons: firstly, chromosome x was not analysis in our study; secondly, the version of sheep reference genome assembly are different, they used ver.
<dig>  from csiro  <cit>  and we used the latest sheep genome release ovis_aries_v <dig>  .
in this study, we found that two windows comprising the ppp1cc and pdgfd genes are associated with significant differences between fat-tail and thin-tail groups.
evidence shows that the first domesticated sheep were thin-tailed and that the fat-tail developed later  <cit> .
as expected, pdgfd was strongly positively selected in fat-tail breeds by the rsb approach.
moreover, the pdgf family promotes proliferation and inhibits differentiation of preadipocytes  <cit> .
in addition, pdgfd is highly expressed in adipose tissue  <cit> .
until now, research on pdgfd has concentrated on its association with diseases, especially cancers in humans.
thus, we hypothesized that pdgfd plays an important role in sheep adipose tissue and is a candidate gene that may lead to the formation of the fat-tail.
CONCLUSIONS
in this study, pca, structure and nj-tree analysis both revealed that chinese sheep populations could be subdivided into two genetic clusters.
one is the tibetan group  and the other is the mongolian and kazakh group .
we suggest that chinese indigenous sheep have descended from two ancestors, from northwest and southwest china, respectively.
we used the di to reveal selection in the sheep populations.
we found known candidate genes such as rxfp <dig>  bmpr1b, msrb <dig>  and kit, mc1r, and fry, which influence horn, lambing percentage, ear size, and coat phenotypes, respectively.
we also detected two strong selection windows that split china sheep into fat-type  and thin-type .
the rsb approach identified a positively selected window that included a candidate gene, pdgfd, for formation of the fat-tail.
further research on the association of this gene with fat deposition in sheep will be performed.

 sheep, primarily raised for meat, wool, milk, and pelts, are an important part of the agricultural economy worldwide. it is the first grazing animal known to have been domesticated. archaeological evidence suggests that sheep were probably first domesticated in the fertile crescent, approximately  years ago. china is one of the nine independent food producers worldwide, and has a long history of sheep husbandry. the earliest record of chinese sheep remains can be traced back to approximately 5000– years ago. china has a great diversity of ecosystems and an abundance of sheep resources. based on geographical distribution and morphological characteristics, there are indigenous sheep breeds in china, which can be classified into three groups: mongolian, kazakh, and tibetan. the native domestic sheep breeds are highly adapted to local environmental conditions, and their most distinctive feature is the type of tail. mongolian sheep are abundant in high latitudes; kazakh sheep have the ability to deposit a large amount of fat in the body to meet nutritional demands during the winter and spring; the tibetan sheep are generally present at low latitudes where it is warmer and experiences less snow. in addition, artificial selection is also an important driving force for the formation of species diversity. most of the chinese domestic sheep are reared for meat, while some varieties are multipurpose. for instance, duolang is an excellent mutton producer, tibetan sheep are one of the major breeds for carpet wool in china, and hu and large-tailed han are prolific lambskin-type breeds. in a previous microsatellite analysis, ma et al. indicated that chinese northern sheep could be divided into two broadly defined lineages, tibetan origin and mongolia origin, representing different geographical clusters. zhong et al. revealed three major clusters of chinese indigenous sheep and pointed out that chinese indigenous sheep have a complicated genetic structure under the effects of different breeding histories, geographical distributions and ecological factors. in the present study, we investigated chinese indigenous breeds that represent the main sheep types in china using the illumina ovine snp genotyping beadchip. our goal was to analyse the population structure and genome selection among chinese native sheep breeds.  genetic variation and population genetic analysis in this study, four metrics were used to estimate levels of within-breed genetic diversity. the polymorphism, expected heterozygosity, observed heterozygosity, and inbreeding coefficients among ten sheep populations were –, –, –, and –, respectively. levels of polymorphism were generally high, more than 92% of loci displaying polymorphism within each population. the values of expected heterozygosity were close to observed heterozygosity in all populations, and the duolang population had the lowest values for heterozygosity. moreover, the he of plateau-type tibetan and valley-type tibetan presented were consistent with previous reports. the lowest inbreeding coefficients were observed in kazakh sheep. we then estimated the ranges of minor allele frequency for all sheep breeds. in all breeds, about 40% of single nucleotide polymorphisms are highly variable and 15% exhibit limited variability. finally, we investigated the extent of linkage disequilibrium, as estimated by the average distance between snps that correspond to different linkage disequilibrium r in each breed. the range of average distance was estimated as –  kb and decreased with the increasing of r value. and the standard deviation was highest when r2 = . to further examine the relationships among individuals, we performed principal components analysis among all individuals. the analysis showed two principal components, with variances of % and %, respectively. according to pc chinese sheep could be divided into two groups consistent with their fat deposition: thin-type sheep and fat-type sheep. they are further subdivided into two different sub-groups distinguished by differences in fat deposition. second, combining the two principal components clustered the three tibetan sheep together and the five mongolian sheep together; however, the two kazakh sheep were clearly segregated. we could distinguish the three breeds in the tibetan group, but it was more difficult in the mongolian group, which was mixed together.figure animals clustered on the basis of principal component analysis using individual genotypes. plot for the first and second component revealed the clustering of animals from uji, hus, ton, lth, lop, kaz, dul, diq, tibp, and tibv breeds; uji: ujimqin sheep, hus: hu sheep, ton: tong sheep, lth: large-tailed han sheep, lop: lop sheep, kaz: kazakh sheep, dul: duolang sheep, diq: diqing sheep, tibp: plateau-type tibetan sheep, and tibv: valley-type tibetan sheep. all pairwise fst values, which were rescaled as fst /, were calculated between the ten populations. the lowest level of differentiation was found between the plateau-type tibetan and valley-type tibetan populations, while the greatest divergence was observed between diqing and duolang. in the mongolian sheep populations, fst between lop and ujimqin was the lowest. in comparison, the pairwise fst within the groups showed a closer relationship than between the groups. we calculated the mean pairwise fst of each breed, which indicated that ujimqin had the lowest mpf and duolang had the largest mpf. the nj-tree showed clearly defined clusters. diqing, plateau-type tibetan, and valley-type tibetan breeds were found in one main branch. this result was well supported by the traditional classification and their close distribution in the neighboring areas of tibetan and qinghai provinces. the other main lineage of the nj tree included the mongolian and kazakh group. in addition, three breeds from xinxiang clustered in the same branch. we also constructed a neighbor-joining tree among the individuals. the results were similar to the nj-tree of the populations and clearly showed that there were no conflicts concerning the origins of individuals assigned to each breed.figure neighbor-joining phylogeny for chinese indigenous sheep breeds based on pairwise f st. uji: ujimqin sheep, hus: hu sheep, ton: tong sheep, lth: large-tailed han sheep, lop: lop sheep, kaz: kazakh sheep, dul: duolang sheep, diq: diqing sheep, tibp: plateau-type tibetan sheep, and tibv: valley-type tibetan sheep. to confirm our observation of the degree of divergence, the program structure was applied to estimate the proportion of common ancestry among the breeds. a model-based unsupervised hierarchical clustering of the individuals was analyzed by considering different k numbers of predefined clusters based on autosomal snps. the results of bayesian clustering for k =  indicated that there was a clear transition from the northwest and north china populations to the southwest and south china populations. we found that fat-type sheep were green dominated, wherein duolang and kazakh accounted for more than 90%. the thin-type sheep were red dominated, and diqing accounted for 90%. this is also consistent with the pca and nj-tree. furthermore, when the k value became large, some breeds were independent. at k =  duolang tended to be separated from the fat-type group. in the pairwise fst analysis, the mean pairwise fst of duolang was the highest among the ten chinese indigenous sheep breeds. thus, duolang might be a population subdivision within the kazakh group. at k =  the kazakh breed separated from the fat-type group. soon after, large-tailed han, hu, and tong separated one after another from the fat-type group when k = 8– figure population structure of sheep inferred by model-based clustering using structure. results from k = are shown. signatures of selection in the ovine genome the di statistic is a new population-genomics strategy based on levels of population differentiation, which determines robustly whether selection acts on newly arisen or pre-existing variations. we performed di statistic analyses to identify candidate regions targeted by selection in chinese indigenous sheep breeds. the di statistic was calculated for autosomal snps in 300-kb windows, with a minimum of three snps per window, and defining the populations by breed. the di statistic is a summation at each window of pairwise fst values for each breed combination, corrected by the value expected from genome-wide calculations; therefore, a large value of di statistic indicates greater divergence at that 300-kb window than that observed across the genome as a whole. in total, snps were evaluated within windows, averaging snps per window. we define candidate selection regions that fell into the upper 99th percentile of the empirical distribution. the windows within each breed were considered putative signatures of selection. in total, of the windows met this criterion in one or more of the breeds. these regions, considered putative signatures of selection in each breed, are listed in additional file 8: table s the maximum di statistic value per breed ranged from in the kazakh to in the valley-type tibetan. additional file 9: figure s shows the genome-wide distribution of the di statistic. to investigate how many selective events were unique or shared among breeds, we calculated the number of overlapping signatures of selection for each of the significant 300-kb windows. sixteen of the significant windows exhibited signatures of selection in four or more breeds. sliding-window analyses of pairwise fst across the 300-kb interval suggested two or more independent selective events, reflected by two peaks of differentiation, with distinct patterns of allele frequency divergence among breeds.figure top strongly selection regions in chinese indigenous sheep. the number of overlapping signatures of selection in each 300-bp window. we defined an overlapping signature of selection for each window if the empirical p value was ≤ in one breed. the black arrow indicates the chromosomal region shown in b and c; the per-snp d i statistic of three regions with three consecutive windows, with t t and t widows in the middle. the braces indicate the group to which the breed belongs; the rsb value of each snp in the three regions, which are consistent with b. the black arrow indicates the group name. the dashed red line denotes the significance threshold at p =. the first peak of differentiated region of the sheep genome showed evidence of selection in multiple breeds on oar. the differentiation observed from to  mb coincides with the rxfp gene as a candidate gene for sheep horns. in the window, we found that the 2nd to 4th snp clearly divided sheep breeds into two groups, one is horned and the other is mixed and polled. the horned breeds showed high levels of differentiation at rxfp compared with the mixed and polled breeds. the second peak of differentiation region in oar from to  mb included the ppp1cc gene, also known as ppp1g, which is a subunit of the protein phosphatase it is a glycogen-associated phosphatase responsible for dephosphorylation and subsequent inactivation of glycogen synthase and is universal in skeletal muscle. ppp1cc is not required for insulin-stimulated glycogen synthesis in skeletal muscle, but appears to be a component of the response to contractile action. recent research indicates that ppp1cc is a positional candidate locus for skeletal muscle strength phenotypes. in the di statistic window, three snps showed higher di statistic values in the thin-type breeds than in the fat-type breeds. we analyzed the remaining significant regions, which showed selection between four and seven breeds, and found the t region in oar which was selected in seven breeds, and divided the breeds into two groups, identical to the t region. this region, from to  mb, included only one functional gene: platelet-derived growth factor d, which is one of the four subfamilies of pdgf, and is a potent stimulator of proliferation. pdgf was first identified and purified from human platelets, where it is sequestered in the alpha granules and released upon platelet activation. studies have shown that the pdgf gene promotes proliferation and inhibits differentiation of preadipocytes. real-time quantitative pcr indicate that pdgfd is expressed at a higher level in adipose tissue e than in human normal tissues, except the thyroid. to better understand the selection of these three regions, they were divided into two groups based on the di statistic value. rsb is a new approach to detect recent positive selection based on contrasting the extended haplotype homozygosity profiles between populations. we calculated the rsb value based on the groups that were included in each window. the rsb results showed region t was selected in horned groups and region t was subject to selection in fat-type populations; however, neither reached statistical significance. interestingly, we found some snps before t and t windows were under significantly selection in rsb analysis. the density of ovine 50 k snp beadchip is not high enough and may lead to low detection ability and deviation. by contrast, region t was significantly positively selected in the fat-type groups and the pdgfd gene promotes proliferation. therefore, we hypothesized that this region may be related to the formation of fat-type sheep. in addition, we also found an interesting peak of differentiation window in oar of six selection breeds, which overlaps with two genes, msrb and lemd a snp in this window, within msrb differentiates duolang strongly from the others. msrb was identified as a candidate gene for ear morphology in dogs and pigs by genome-wide association studies. at this locus, duolang and diqing show the two extremes of the allele frequencies. from the pictures of each breed, we observed that the ears of the duolang sheep are the largest of all the sheep breeds. figure 5c shows photographs of the two extreme breeds. therefore, we hypothesized that that this snp is associated with ear size in sheep.table annotation of the top six signatures of selection windows frequency breed region annotated gene eef1a rxfp2 muted,ppp1cc pdgfd msrb lemd3 nf omg, evi2b aasdhppt,ankrd gpr lrrc mre11a breed abbreviations are described in table  breeds included in the study and their distinguishing phenotypes breed location group tail type horn type coat color breeding objective lambing percentage all information comes from animal genetic resources in china - sheep and goats. a snp associated with sheep’s ear morphology. the per-snp d i statistic in –  mb on oar the black arrow indicates a snp that is associated with ear size; the allele frequencies of chinese indigenous sheep breeds; red squares represents a and green squares represent g; duolang and diqing: two extremes of the allele frequencies. cross traces in duolang duolang is a special breed that showed a large genetic distance from other chinese indigenous breed in a previous population genetic analysis. historically, a religious person brought jill wagner sheep from afghanistan to xinjiang in and then crossed them with native sheep breeds, eventually forming a new breed called duolang. the major characteristics are large body size, fast growth and a high reproductive rate. in the present study, we found two consecutive selection regions on oar and involving a total of windows, which were almost half of the candidate selection regions of duolang. these windows contained candidate genes. in oar, the highest di statistic window did not include any genes, and the second highest di statistic window included nine genes. npr which was found by both kijas and moradi, is involved in skeletal morphology and body size. spag and hint influence carcass weight and birth body weight, respectively. in oar, we found that the highest di statistic window was for a region associated with a milk production qtl in cattle, which included four genes, mepe and ibsp). in particular, spp is a candidate gene for the litter size in pig.figure plot of d i windows of two regions on oar and oar of chinese indigenous sheep breeds. a black arrow indicates the peak windows in each region. the braces include the candidate genes. other important candidate genes selected in less than three breeds such universal differentiation at a single locus is consistent with the action of a gene that generally sorts individuals into phenotypic classes and breed groups. in this study, we also found some common selection genes that influence reproduction and coat color. bmpr1b, located on oar, showed strong evidence of selection in highly prolific breeds hu and large-tail han. although bmpr1b is associated with high prolificacy in some chinese breeds or strains of sheep, other researchers believe that bmpr1b is not the only gene responsible for the fecundity of chinese sheep. we also identified that kit, mc1r, and fry influence coat color. a region on oar that includes the kit gene is associated with melanocyte development and accounts for pigmentation phenotypes in pigs and horses, which also shows a strong signature of selection in the duolang and kazakh breeds, both of which have dark coat colors. mc1r was selected in the ujimqin population. a mutation in mc1r causes black spotting in pigs. fry, which is a key candidate gene involved in the piebald phenotype in merino sheep, is located on chromosome and was selected in valley-type tibetan sheep, whose coat has black spots. interestingly, fry was also considered as a candidate gene affecting wool quality between rambouillet and suffolk sheep. furthermore, wnt and wnt10a, which were only selected in the rumped tail breeds kazakh and duolang, inhibit adipogenesis via a β-catenin-dependent mechanism. discussion in the present study, population genetic analysis was performed on 50 k snp genotypes of animals for chinese indigenous sheep breeds. we analyzed the population using pca, structure, and nj-tree. the results indicated that chinese indigenous sheep populations could be subdivided into two genetic clusters: the tibetan group and the mongolian and kazakh group. overall, the partitioning of genetic diversities of the breeds is consistent with their geographic distributions. the mongolian group is the most widely distributed breed in china, mainly in inner mongolia, the central plains, and eastern coastal areas. this is attributed to good adaptability and performance of the mongolian sheep and genghis khan’s expedition in the yuan dynasty. the most abundant species diversity is in xinjiang province, which is mainly attributed to its location. it is the only way to the trading venues in the “silk road”, and its position in central asia and the central plains, and the geographic isolation of its southern and northern parts is one reason for its rich diversity. the tibetan group living in the southwest region, which is a mountainous region, also has abundant genetic diversity. according to pairwise fst, the relationships between lop and ujimqin and between plateau-type tibetan and valley-type tibetan were closer than others. these two combinations were observed to be mixed together in the pca plot, and had a similar composition in structure from k = 2–. the greatest divergence was observed between diqing and duolang, which come from different groups: diqing belongs to the tibetan group and duolang belongs to the kazakh group. a previous study indicated that the mongolian group was the origin of tong sheep. however, according to our study, ujimqin is the oldest breed in the mongolian population compared with tong sheep, because ujimqin not only has the smallest mpf and the highest polymorphism in the mongolian group, but also it lives closer to mongolia, the capital of the yuan dynasty, geographically. population genetic analysis was able to distinguish physiological differences and geographical origins. in this study, duolang and kazakh come from xinjiang province; however, their genetic difference was in the middle of the range of differentiation, and similar results were seen from pca and structure. one reason is that the tianshan mountains divide xinjiang into southern and northern regions: duolang comes from the southern region and kazakh comes from the northern region. this results in the geographical isolation between duolang and kazakh. there is another explanation, according to record, duolang was mixed with afghanistan sheep, which may be the cause of this phenomenon. interestingly, we found lop, duolang, and kazakh clustered in a branch of the nj-tree. however, lop belongs to the mongolian group, according to the pca figures it is closer to mongolian sheep breeds than duolang and kazakh. in structure result when k = 4– lop breed appears to be some introgressed of kazakh group, but this needs more intensive research to explain. these data provided strong evidence of the genetic structure in these sheep breeds. in this study, which involved just a quarter of chinese local breeds, there were very complex genetic relationships. for more accurate understanding of the relationships between them, more breeds should be analyzed. selection in chinese sheep breeds most studies of artificial selection in sheep have focused on single-gene analyses arising from phenotype-driven studies. recently, kijas et al. analyzed sheep breeds worldwide, one of which was a chinese native breed, tibetan sheep. the study identified the strongest selected candidate gene, rxfp in response to breeding for the absence of horns. we also identified this gene in the peak of the differentiated region in the chinese population. most chinese indigenous sheep are dual-purpose, low breeding breeds. only a few specialized breeds are used for lambskin and lamb fur. hu, large-tailed han and tong are lambskin breed. hu and large-tailed han are amongst the most prolific breeds in the world. bmpr1b, a notable candidate gene, was selected in both breeds. other candidate genes focused on profile characteristics, such as kit, mc1r, and fry, which influence variation in dark coat color and the piebald phenotype. the fat-tail is an important component in sheep. currently, approximately 25% of the world sheep population comprises fat-tail breeds, which are found in a wide range of countries in asia, the middle east and north africa. in china, fat-tail breeds comprise 80% of the population. in terms of tail type, chinese breeds are divided into four groups: short fat-tailed, long fat-tailed, fat-rumped, and thin-tailed. the first three types of tail shape are not the same, but still belong to fat-type sheep. this trait is now commercially less important because of improved forage availability and decreased price of the product. therefore, a decrease in fat-tail size is often desirable for chinese producers. to date, several investigations into the inheritance of fat-tails have been undertaken. moradi et al. confirmed three regions located on chromosomes and x that affect thin and fat tail breeds. bakhtiarizadeh et al. suggested that the expression of fabp in the fat-tail is an important index of fat deposition. the haplotype in cast can distinguish between fat-tailed and thin-tailed sheep breeds. recently, wang et al. identified genes that were differentially expressed between fat-tailed and thin-tailed groups using rna-seq, and identified nell and fmo as genes relevant to fat metabolism in adipose tissues. despite these studies, there is no compelling reason for the impact of a fat-tail on the formation mechanism. it is worth mentioning that the results of moradi’s and our are inconsistent, even if both of us used similar type of population and the ovine 50 k snp bead chip. there include two reasons: firstly, chromosome x was not analysis in our study; secondly, the version of sheep reference genome assembly are different, they used ver. from csiro and we used the latest sheep genome release ovis_aries_v. in this study, we found that two windows comprising the ppp1cc and pdgfd genes are associated with significant differences between fat-tail and thin-tail groups. evidence shows that the first domesticated sheep were thin-tailed and that the fat-tail developed later. as expected, pdgfd was strongly positively selected in fat-tail breeds by the rsb approach. moreover, the pdgf family promotes proliferation and inhibits differentiation of preadipocytes. in addition, pdgfd is highly expressed in adipose tissue. until now, research on pdgfd has concentrated on its association with diseases, especially cancers in humans. thus, we hypothesized that pdgfd plays an important role in sheep adipose tissue and is a candidate gene that may lead to the formation of the fat-tail.  in this study, pca, structure and nj-tree analysis both revealed that chinese sheep populations could be subdivided into two genetic clusters. one is the tibetan group and the other is the mongolian and kazakh group. we suggest that chinese indigenous sheep have descended from two ancestors, from northwest and southwest china, respectively. we used the di to reveal selection in the sheep populations. we found known candidate genes such as rxfp bmpr1b, msrb and kit, mc1r, and fry, which influence horn, lambing percentage, ear size, and coat phenotypes, respectively. we also detected two strong selection windows that split china sheep into fat-type and thin-type. the rsb approach identified a positively selected window that included a candidate gene, pdgfd, for formation of the fat-tail. further research on the association of this gene with fat deposition in sheep will be performed. 
in pca plot, the tibetan and mongolian groups were clustered as expected; however, duolang and kazakh were segregated. based on the results presented here, we propose that chinese native sheep can be divided into two genetic groups: the thin type, and the fat type. herein, we aimed to evaluate the population structure and genome selection among individuals from ten representative chinese indigenous sheep breeds: ujimqin, hu, tong, large-tailed han and lop breed; duolang and kazakh; and diqing, plateau-type tibetan, and valley-type tibetan breed. we analyzed the population using principal component analysis, structure and a neighbor-joining -tree. in the nj-tree, the tibetan group formed an independent branch and the kazakh and mongolian groups were mixed. we also identified known candidate genes such as bmpr1b, msrb and three genes that influence lambing percentage, ear size and coat phenotypes, respectively. we also identified important genes that drive valuable phenotypes in chinese indigenous sheep, especially pdgfd, which may influence fat deposition in fat type sheep. two consecutive selection regions in duolang sheep were obviously different to other breeds. traditionally, chinese indigenous sheep were classified geographically and morphologically into three groups: mongolian, kazakh and tibetan. we then used the di statistic approach to reveal selection in chinese indigenous sheep breeds. we detected three strong selection windows involving three functional genes: rxfp ppp1cc and pdgfd. pdgfd, one of the four subfamilies of pdgf, which promotes proliferation and inhibits differentiation of preadipocytes, was significantly selected in fat type breeds by the rsb approach.
 traditionally, chinese indigenous sheep were classified geographically and morphologically into three groups: mongolian, kazakh and tibetan. herein, we aimed to evaluate the population structure and genome selection among individuals from ten representative chinese indigenous sheep breeds: ujimqin, hu, tong, large-tailed han and lop breed; duolang and kazakh; and diqing, plateau-type tibetan, and valley-type tibetan breed.  we analyzed the population using principal component analysis, structure and a neighbor-joining -tree. in pca plot, the tibetan and mongolian groups were clustered as expected; however, duolang and kazakh were segregated. structure analyses suggested two subpopulations: one from north china and the other from the southwest. in the nj-tree, the tibetan group formed an independent branch and the kazakh and mongolian groups were mixed. we then used the di statistic approach to reveal selection in chinese indigenous sheep breeds. among the genome sequence windows analyzed, sixteen exhibited signatures of selection in four or more breeds. we detected three strong selection windows involving three functional genes: rxfp ppp1cc and pdgfd. pdgfd, one of the four subfamilies of pdgf, which promotes proliferation and inhibits differentiation of preadipocytes, was significantly selected in fat type breeds by the rsb approach. two consecutive selection regions in duolang sheep were obviously different to other breeds. one region was in oar including three genes the influence growth traits. the other region was in oar including four genes associated with a milk production quantitative trait locus. we also identified known candidate genes such as bmpr1b, msrb and three genes that influence lambing percentage, ear size and coat phenotypes, respectively.  based on the results presented here, we propose that chinese native sheep can be divided into two genetic groups: the thin type, and the fat type. we also identified important genes that drive valuable phenotypes in chinese indigenous sheep, especially pdgfd, which may influence fat deposition in fat type sheep. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords genome-wide analysischina indigenous sheeppopulation analysisselection signal analysisissue-copyright-statement© the author 2015 