BACKGROUND
copy number variation , as a form of widespread genomic structural variations, has been reported in many model organisms, such as primates and rodents
.
compared with single nucleotide polymorphisms , cnvs seem to have a stronger impact on phenotype and are shown to have effects on changes in gene expression levels
<cit> , which can be explained by disruption of gene dosage, unmasking of recessive alleles, and loss of regulatory elements or regulatory polymorphisms
<cit> .
several recent publications have reviewed the effects of cnvs on gene expression and human diseases
.
in addition, cnv provides materials and mechanisms for creating new genes
<cit> .
given the importance of cnvs and their high rates of mutation, interest in cnv detection has extended to domesticated animals, including dogs
<cit> , pigs
<cit> , goats
<cit> , horse
<cit> , and sheep
<cit> .
similarly, cnv and copy number variable regions  have been a hot-spot in cattle genomic variation researches, which may be associated with, or affect, cattle’s health and production traits under recent selection.
previous studies have produced several cnv datasets on cattle
.
some are focused on one single breed by using snp array: such as, bos taurus coreanae  and chinese holstein cattle .
other cnv evidences for multiple breeds are also shown on snp array.
for example, matukumalli et al.
identified  <dig> candidate deletions by using an earlier version of bovinesnp <dig> assay
<cit> , and hou et al.
found  <dig> candidate cnvrs in  <dig> modern cattle breeds and  <dig> out-groups
<cit> .
at the same time, more studies are conducted on microarray-based comparative genomic hybridization :  <dig> high-confidence cnvrs in  <dig> breeds
<cit> , and  <dig> cnvrs in  <dig> breeds
<cit> .
most recently, the next-generation sequencing was also used to detect cnvr with more power
<cit> .
and in these studies, the researchers focus more on the detection of cnv in different breeds.
up to this date, few studies have confirmed the genome-wide presence of cnvs in chinese native cattle breeds.
compared to the previous cnv investigations mostly focusing on cnv detection, here we selected  <dig> breeds in three main bovine groups in china  to conduct a genome-wide cnv analysis and further examined their effects on gene expression and growth traits of cattle.
overall, we got started with genome-wide cnv screening of three cattle groups, and further associated them with cattle gene expression and body measurements, which provides novel insights into understanding the role of cnv in genomic variation studies.
methods
sample collection
for cgh analysis, we collected blood samples all over china in  <dig> bovine breeds or populations: twelve b. taurus breeds : anxi, bohaihei, chinese holstein, jiaxian, jinnan, leiqiong, luxi, mongolia, nanyang, qinchuan, wannan and zaosheng; one b. grunniens : tianzhu white yak; and two b. bubalis ones : swamp buffalo and river buffalo .
diverse tissues of fetal, calf  and adult  in qinchuan breed were collected in the slaughter house for gene expression analysis .
blood samples of nanyang , jiaxian  and qinchuan  were collected with body measurements , including body height, body length, heart girth, hucklebone width, and body weight for association analysis.
all our sample collection was carried out in strict accordance with the ethical guidelines approved by the animal care commission of college of animal science and technology, northwest a & f university.
genomic dna was extracted and purified from whole blood following standard procedures
<cit>  and quantified by spectrophotometry and agarose gel electrophoresis.
total rna was isolated from flash-frozen tissues.
first-strand cdna was synthesized from  <dig> ng of total rna with the prime script rt reagent kit  according to the manufacturer’s instructions.
array cgh platform
we quantified copy number by hybridizing dna to nimblegen3x720k cgh array , which provided an evenly distributed coverage of ~ <dig>  oligonucleotide probes .
the probes of 50–75 bp in length were designed with similar melting temperatures based on btau_ <dig>  genome assembly
<cit> .
we chose one pure-blooded angus bull as the reference.
dna labelling, hybridization, washing, array scanning, and array imaging were carried out according to the previously described
<cit> .
briefly, pairs of genomic dna  were labeled with fluorescent dyes cy <dig>  or cy <dig> , and were co-hybridized on hybridization platform.
the arrays were scanned and fluorescent intensity raw data was extracted.
the initial data analysis  was performed on nimblescan v <dig>  software with segmnt algorithm .
we used an updated version of the previously described method to do cnv calling, ie., determining copy number gains and losses by changes in log <dig> signal intensity
<cit> .
the segment, with mean log <dig> ratio ≥ | <dig> | and at least  <dig> consecutive probes covered, was defined as a cnv.
cnvrs in one group were determined by aggregating overlapped cnvs of all samples
<cit> .
cattle gene annotations were downloaded from the ucsc genome browser , and cattle quantitative trait loci
<cit>  were from the animal qtl database .
the genome positions were converted among genome assemblies of btau_ <dig> , btau_ <dig> , and btau_ <dig> .
<dig> by using the ucsc binary software liftover.
we wrote perl scripts to search for gene content and quantitative trait loci  inside cnvrs, and determined a positive gene/qtl by > 50% overlap.
gene ontology  identifiers were retrieved with refgene ids in r package biomart and plotted by the web histogram tool wego
<cit> .
cnvr chromosome plotting, clustering analysis, nonmetric multidimensional scaling , go identifier retrieving, and indicator species analysis  were performed by using r packages of ggbio, pvclust, vegan, biomart, and indicspecies, repectively.
principal component analysis  was performed in stamp v <dig>
<cit> .
data access
raw array cgh data in this study has been deposited in ncbi geo database under accession number of gse <dig>
quantitative pcr
we performed a qpcr analysis to validate copy number changes detected by array cgh based on the relative comparative cycle threshold  method.
primers  were designed by using beacon designer™ .
pcr reaction was done in a volume of 20 μl containing 20 ng of genomic dna,  <dig>  μm of each primer, and sybr premix ex taq™ ii reagents .
analysis of resultant crossing thresholds  was performed based on the ΔΔct method
<cit> , and ΔΔct values were determined by comparing test samples and angus reference  with btf <dig> gene as internal control.
finally, the relative copy number for each sample was calculated as 2-ΔΔct.
we selected two qpcr-confirmed cnvr <dig> and cnvr <dig> for further analysis.
first, we used the primers  to determine cnvr types  in  <dig> individuals against the angus reference.
then we selected checked individuals  for gene expression analysis of pla2g2d in cnvr <dig> and myh <dig> in cnvr <dig> on the cfx- <dig> real-time pcr detection system .
two primer pairs were used for expression detection of pla2g2d and myh3: pla2-f, 5′-gacatactggacctgaac-3′; pla2-r, 5′- agccatagtgtgaatagaag-3′; myh3-f, 5′-agtcgtcagttggaggaa-3′; myh3-r, 5′-gctcttctatttgctgggtaa-3′.
gapdh gene was used for normalization.
the reaction was performed in a volume of 25 μl, containing  <dig>  μl sybr premix ex taq ii, 1 μl of each primer , 2 μl cdna , and  <dig>  μl h2o.
the relative fold change was calculated using 2-ΔΔct <cit> .
mean expression levels and standard deviations were obtained by repeating three independent experiments.
association analysis between cnvr types and growth traits
we evaluated all kinds of factors, and selected three major ones of farm, genotype and breed to build a reduced adjusted linear model:  <dig>
where yijk is trait measurement, μ is overall population mean, fi is farm, gj is genotype effect, bk is breed, and eijk is random error.
we used the least-squares means  to estimate the association between cnvr types and body measurements in spss software
<cit> .
RESULTS
cnvrs in cattle groups
in taurine, we identified  <dig> cnvrs covering the region of 47 mb on the placed chromosomes , together with  <dig> cnvrs on chrunall .
all  <dig> cnvrs correspond to  <dig> % of the bovine genome , which consist of  <dig> loss,  <dig> gain and  <dig> both  events .
loss events are approximately 3-fold more than gain ones, while both ones are much longer than the others on average.
furthermore,  <dig> cnvrs are only found in one individual, and  <dig> cnvrs are shared in two or more ones, among which  <dig> multiple events have a frequency of ≥  <dig> .
in yak/buffalo groups, we identified 161/ <dig> candidate cnvrs, which consist of 123/ <dig> loss, 34/ <dig> gain, and 4/ <dig> both events.
duplications provide additional copies of genes, and this kind of redundancy can allow more flexibilities of gene loss by selective pressure.
mutation and selection can result in functional changes introducing a new function or specialization of old functions
<cit> .
thus, gain events might be the first step for extra genetic material during cattle breed formation, and their followed isolated genome cnv evolution and adaption may be a potent evolutionary force for more loss events.table  <dig>
cnvr summary for chinese bulls
*percentage of total cnvr length in the genome assembly of btau_ <dig> .
“avg” is for cnvrs per sample.
genomic distribution of cnvrs in chinese bulls.
<dig> cnvrs  in  <dig> bulls are distributed across chromosomes, in which the above are for taurine , while below are the cnvrs for yak  and buffalo .
refgenes from ucsc genome browser are shown inside the chromosomes in black.
on combining three groups, we identified  <dig> cnvrs totally,  <dig> % of the bovine genome, among which  <dig> ones in yak  and  <dig> ones in buffalo  are shared by taurine , and about  <dig> cnvrs are overlapped in all three groups .
we then compared  <dig> shared cnvrs on the placed chromosomes to those in previous studies , and confirmed them all, except for cnvr <dig> and cnvr <dig>  which indicated their reliability.
it is notable that cnvr frequencies in different studies are shown diverse, and more shared cnvrs are detected based on cgh and re-sequencing methodologies than that of snp-array-based studies .
and cattle breeds, which may have experienced different selection pressures, contribute a lot to cnvr differences as well.
it concludes that samples and platform may have the greatest effects on cnv detection.figure  <dig>
venn diagram of cnvrs in three groups .
cnvrs are overlapped among taurine, yak, and buffalo in the specific area.
cnvrs shared by this study and other studies on cattle cnv
*shared cnvrs do not contain the ones on chrunall.
we performed an integrated hybridization on the three bovines in one cgh platform, and gained an advantage of parallel comparison, regardless of how different their genomes are.
our array probes were designed based on genome assembly btau_ <dig>
<cit> .
yak genome had been released after our cgh experiment was done, and shown very similar to that of cow
<cit> .
up to date, the complete genome of buffalo is not placed well in ncbi, and only unplaced sequences are ready to download.
all our cnvrs in yak and buffalo are from the orthologous regions of cow genome, and our  <dig> shared cnvrs are supposed to be from the common orthologs of the three bovine genomes.
however, their genome positions in yak and buffalo may be different and should be considered cautiously.
and the yak- and buffalo-specific regions are not covered by our probes, and out of our scope.
it is noted that the bovine cgh array had also been used to scan cnv of goat and sheep
<cit> .
in addition, we designed five probes covering the mitochondrial region.
although there is only one cnvr in chrm in three groups, we found a difference between taurine  and yak  & buffalo  .
the mitochondrion dna copy number varies referring to energetic metabolism among cell types
<cit>  and fertility between oocytes and sperm
<cit> .
multiple copies of mtdna in the same mitochondrion are directly associated with the amount of atp synthesized
<cit> .
our finding on mtdna copy number difference suggests possible low energetic metabolism in yak and buffalo’s blood, and more studies are necessary to understand the role of mitochondrial copy number in cattle’s traits and performances.
we had discovered cnvrs’ distribution preferences across chromosomes.
just like the previous results
<cit> , cattle cnvs are distributed in a non-random way in this study, and their contents vary across chromosomes.
the proportion of any known chromosome susceptible to cnvrs ranges from  <dig>  to  <dig> % , although chrunall shows the strongest enrichment of cnvrs , probably due to highly repetitive sequences in these unplaced contigs.
except for chrm, we got strong cnv-enriched chromosomes of  <dig>   <dig>   <dig>   <dig>   <dig>  × and  <dig> , which might have been shaped by local chinese bulls, compared with other results
<cit> .
clustered cattle cnvrs
selection has also been shown to shape the architecture of segmental duplications during human genome evolution
<cit> , and studying cnvs’ evolution may help us reveal the genomic alteration and environmental driving impact.
the cluster analysis of cnvr in cattle and pig had evidenced that cnvr loci are consistent with the breed divergence and history
<cit> .
so we performed a clustering analysis of cnvrs on all individuals, which revealed remarkable profiles among groups.
first, the three groups of taurine, yak, and buffalo are clearly divided .
second, individuals in one single breed are easily clustered closely.
the clustering results simply showed a phylogeny, while the principal component analysis  results showed their detailed relations.
the pca plotted samples into three groups .
similarly, we investigated the impact of groups on cnvrs structure by using nonmetric multidimensional scaling , which is to visualize the interrelationships among a complex dataset and level of similarity of individuals, and generally grouped samples into taurine, yak, and buffalo .
the results supported the hypothesis that genome structure variations, especially cnvs, may be raised by isolated evolutions and shaped by breed formation and adaptation
<cit> .
to highlight potential evolutionary contributions of cnvs to chinese major cattle breeds’ formation and adaptation, we identified  <dig> cnvrs which are abundant statistically in three groups  by using indicator species analysis
<cit> .
compared to  <dig> shared cnvrs from the orthologs, the biased ones reflect their unique genomic backgrounds, which suggest a potential variance of the three bovine genomes.
our parallel comparisons based on one single cgh platform are reliable, which overcome the shortcoming that it is difficult to compare the datasets by different technologies and methods.
gene content and quantitative trait loci  in cnvrs
totally  <dig> cnvrs encompass  <dig> genes, which are shown with refgene id and gene name .
in order to determine biological functions of copy number variable genes, a gene ontology  analysis annotated  <dig> out of  <dig> genes in three main go categories: cellular component, molecular function and biological process .
as shown in the go map, genes in all categories were mainly involved in eight ones, including cell/cell part , organelle , binding , catalytic activity , metabolic process , cellular process , pigmentation , and biological regulation .
this set of copy number variable genes possesses a wide spectrum of molecular functions, and provides a rich resource for hypotheses on their genetic basis of phenotypic variation within and among breeds.
many cattle specific genes were also found in our cnvrs, such as c-type lysozymes, bsp30a, interferon tau subfamilies, wc <dig>  and itln <dig> <cit> .
moreover, eda gene, which has been reported a deletion and responsible for hypotrichosis and dental defects in cattle
<cit> , and slc4a <dig> gene, which is a copy number variable gene and association with osteopetrosis, are both confirmed in our study
<cit> .
we also downloaded  <dig> cattle qtls from animal qtls database, and searched for potential qtls which reside inside  <dig> cnvrs on placed chromosomes .
there are  <dig> %  of cnvrs overlapped with  <dig> qtls.
the qtls in multiple cnvrs are associated with exterior , health , meat and carcass , milk , production  and reproduction .
the results are in accordance with chinese breeding history.
cnvr confirmation and effects on gene expression
to evaluate the accuracy of copy number assignments, quantitative real time-pcr was used as described previously
<cit> .
we selected nine detected cnvr, including loss, gain, and both types, whose frequencies range from  <dig> % to  <dig> %.
the selected cnvrs were all overlapped with genes and qtls of meat, carcass and production .
totally  <dig> pairs of primers were used, one or two pairs to cover one selected cnvr.
the results showed that out of  <dig> qpcr assays,  <dig> ones  confirm the predictions by array cgh.
false-positive identification is common in cnv detection, and there is always to some extent <  <dig> of confirming rate
<cit> .
in fact, cnvrs were of complex structure, and qpcr can only target a small portion, which does not reflect their complete characteristics.
and the boundaries of cnvr by arrays are indistinct.
notably the average size of  <dig> confirmed cnvrs is  <dig>  kb, much smaller than those of three unconfirmed ones .
certain number of samples are chosen randomly as negative control for the reliability of results
<cit> .
in rats, only 44% genes in cnvrs are differentially expressed
<cit> .
we selected two genes  for detailed exploration of expression levels, because both of them might have effects on the performances of cattle.
pla2g2d is an innate immunity gene, and thought to play a role in gonadotropin-releasing hormone and mark signaling
<cit> , which had also been identified inside a cnvr of black angus by re-sequencing method
<cit> ; most recently, it was confirmed that the copy numbers of pla2g2d gene were associated with the index of total merit in holstein bulls
<cit> .
the findings were very important because it was the direct evidence of complex traits of livestock which may be modulated in part by cnvs.
and myh <dig> is expressed mainly in embryo and muscle
<cit> , and its mutation caused the freeman-sheldon and sheldon-hall syndrome
<cit> .
in cattle, the snp in myh <dig> was also associated with the growth and carcass traits in chinese qinchuan cattle
<cit> .
so, we firstly examined the expression profiles of both genes in qinchuan cattle.
the results showed that mrna of pla2g2d was mainly expressed in spleen, intestine, adipose, and lung , while myh <dig> mrna was primarily expressed in fetal muscle, and liver, spleen and muscle of calf .
secondly, considering both tissue importance in growth traits and mrna expression, we selected  <dig> samples of adult adipose tissues  and  <dig> ones of calf muscle tissues  to analyze cnvr’s effects on mrna of pla2g2d and myh <dig>  respectively.
the results revealed a significant negative correlation between mrna levels of pla2g2d and cnvr <dig> .
the correlation could be due to position effect of cnvr, and a regulatory sequence of pla2g2d may exactly reside in cnvr <dig>
<cit> .
interestingly, the regulatory sequence may be a transcriptional upper repressor and suppressed pla2g2d mrna expression.
however, we have not seen any evidence of correlation between myh <dig> transcript expression and cnvr <dig>  it is well known that the break point definition of cnvrs by arrays is equivocal and only  <dig> % of myh <dig> gene is overlapped with cnvr <dig>  so it is possible that myh <dig> or its regulatory motif was not covered by cnvr <dig>  it is also worth emphasizing that the dosage compensation, lack of regulatory elements in the duplicated copy, differences in the chromatin environment and many other factors might keep mrna levels stable
<cit> .figure  <dig>
relative mrna expression level of
pla2g2d
in cnvr <dig> in adipose tissues.
relative pla2g2d mrna expression levels in adipose tissues of  <dig> selected qinchuan cattle individuals  were analyzed by qpcr, and normalized against that of gapdh.
cnv types were determined against the angus reference, and the normal type means the same to the reference.
three independent experiments were repeated for reliability.
an asterisk denotes a significant difference by t-test .
cnvs’ association with growth traits of cattle
cnvs may affect phenotype by altering transcriptional level of genes within or adjacent to cnvr and subsequently alter translation levels
<cit> .
the association between the cnvs and production traits of economic interest had been reported.
in swine, several copy number variable genes were identified as candidate genes for phenotypes related to carcass length, backfat thickness, abdominal fat weight, length of scapular, intermuscle fat content of logissimus muscle, body weight at 240 day, and fatness
<cit> .
in cattle, the direct evidence of close associations of cnvr# <dig> with index of total merit and genetic evaluations for protein production, fat production, and herd life in holstein had been identified
<cit> .
we had demonstrated that the genes inside cnvrs might be expressed differently, and many cnvrs also overlapped with qtls which are associated with cattle performance.
so we believed that cnvrs should be potentially associated with cattle body measurements.
here we collected  <dig> chinese cattle samples in qinchuan, jiaxian and nanyang breeds , and evaluated the associations between cnv types and growth traits  in equation  <dig>  the results indicate that heart girth and body length are significantly associated with cnv types in cnvr <dig> .
individuals of loss type have larger heart girth and hucklebone width .
the location of cnvr <dig> on chromosome  <dig> exactly fell into qtls with various functions, including qtls  <dig> and  <dig> for production , qtls  <dig>  and  <dig>  for meat and carcass
<cit>  in beef cattle .
furthermore, pla2g2d gene was overlapped with cnvr <dig>  cattle of loss type in cnvr <dig> with higher pla2g2d expression maybe finally gain more fat deposition.
in addition, association between cnv of pla2g2d gene and index of total merit had been reported in holstein
<cit> .table  <dig>
association analysis of cnvr <dig> with body measurements
*lsm ± se, “lsm” for least squares mean, “se” for standard error.
“n” indicates sample number.
a, bmeans with different superscripts were significantly different .
on the other hand, we also found that cnvr <dig> is significantly associated with heart girth , probably due to the fact that cnvr <dig> was overlapped with the qtls for production  and meat and carcass  in beef cattle .
regardless of one single gene’s contributions, we had a direct look at the effects of selected cnvrs on cattle body performances, which might be caused by a group of genes.
it took gene population effects into consideration, rather than focusing on only one single trait-related gene.
the association between cnvs and traits is inspiring, but larger population may be needed to validate it.
CONCLUSIONS
we have performed a comprehensive genomic analysis of cnvs based on cgh arrays in chinese cattle, and a detailed functional investigation for cnvrs’ effects on both gene expression and cattle body measurements.
we identified  <dig> cnvrs in b. taurus, which covered  <dig> % of the bovine genome, together with  <dig> and  <dig> cnvrs in b. grunnies and b. bubalis.
furthermore, we confirmed that cnvr <dig> had significantly negative effects on both pla2g2d gene expression and cattle body measurements, while cnvr <dig> showed a significant negative association with heart girth.
our results generated a valuable genome-wide variation resource for chinese cattle genomic researches, and provided a novel insight into understanding the association between animal complex traits and cnvrs during their adapting to local geographical environment and domesticated needs from human society.
electronic supplementary material
additional file 1: table s <dig> : the breeds or populations of chinese cattle used in the cgh arrays.
table s <dig> .
the breeds or populations of chinese cattle used in other experiments.
table s <dig>  primer sequences and results for qpcr.
table s <dig>  cnvrs in chinese bulls.
table s <dig>  shared cnvrs by this study and other studies.
table s <dig>  cnvr in chrm in chinese bulls.
table s <dig>  cnv types of individuals on cnvr <dig> and cnvr <dig> in three chinese breeds.
table s <dig>  association analysis of cnvr <dig> with body measurements.
additional file 2: figure s1: clustering results of cnvrs.
figure s <dig>  pca and nmds of cnvrs on chinese bulls.
figure s <dig>  gene ontology  annotations for genes covered by cnvrs.
figure s <dig>  expression pattern analysis of pla2g2d gene and myh <dig> gene.
abbreviations
cnvcopy number variations
cnvrcopy number variable region
cghcomparative genomic hybridization
gogene ontology
myh3myosin- <dig>  b. taurus myosin, heavy chain 3
isaindicator species analysis
pla2g2db.
taurus calcium-dependent phospholipase a2
nmdsnonmetric multidimensional scaling
pcaprincipal component analysis
qpcrquantitative real time pcr
qtlsquantitative trait loci
snpsingle nucleotide polymorphism.
competing interests
the authors declared that they have no competing interests.
authors’ contributions
lzz and hc conceived and designed the experiments.
lzz, mjy and yx performed the experiments.
lzz and sgj analyzed the data.
czl, xyl, clz, jjs, yzh, yz, cjl, and xz contributed reagents/materials/analysis tools.
lzz and sgj wrote and revised the manuscript.
all authors read and approved the final manuscript.

 copy number variation, as a form of widespread genomic structural variations, has been reported in many model organisms, such as primates and rodents. compared with single nucleotide polymorphisms, cnvs seem to have a stronger impact on phenotype and are shown to have effects on changes in gene expression levels, which can be explained by disruption of gene dosage, unmasking of recessive alleles, and loss of regulatory elements or regulatory polymorphisms. several recent publications have reviewed the effects of cnvs on gene expression and human diseases. in addition, cnv provides materials and mechanisms for creating new genes. given the importance of cnvs and their high rates of mutation, interest in cnv detection has extended to domesticated animals, including dogs, pigs, goats, horse, and sheep. similarly, cnv and copy number variable regions have been a hot-spot in cattle genomic variation researches, which may be associated with, or affect, cattle’s health and production traits under recent selection. previous studies have produced several cnv datasets on cattle. some are focused on one single breed by using snp array: such as, bos taurus coreanae and chinese holstein cattle. other cnv evidences for multiple breeds are also shown on snp array. for example, matukumalli et al. identified candidate deletions by using an earlier version of bovinesnp assay, and hou et al. found candidate cnvrs in modern cattle breeds and out-groups. at the same time, more studies are conducted on microarray-based comparative genomic hybridization: high-confidence cnvrs in breeds, and cnvrs in breeds. most recently, the next-generation sequencing was also used to detect cnvr with more power. and in these studies, the researchers focus more on the detection of cnv in different breeds. up to this date, few studies have confirmed the genome-wide presence of cnvs in chinese native cattle breeds. compared to the previous cnv investigations mostly focusing on cnv detection, here we selected breeds in three main bovine groups in china to conduct a genome-wide cnv analysis and further examined their effects on gene expression and growth traits of cattle. overall, we got started with genome-wide cnv screening of three cattle groups, and further associated them with cattle gene expression and body measurements, which provides novel insights into understanding the role of cnv in genomic variation studies. methods sample collection for cgh analysis, we collected blood samples all over china in bovine breeds or populations: twelve b. taurus breeds: anxi, bohaihei, chinese holstein, jiaxian, jinnan, leiqiong, luxi, mongolia, nanyang, qinchuan, wannan and zaosheng; one b. grunniens: tianzhu white yak; and two b. bubalis ones: swamp buffalo and river buffalo. diverse tissues of fetal, calf and adult in qinchuan breed were collected in the slaughter house for gene expression analysis. blood samples of nanyang, jiaxian and qinchuan were collected with body measurements, including body height, body length, heart girth, hucklebone width, and body weight for association analysis. all our sample collection was carried out in strict accordance with the ethical guidelines approved by the animal care commission of college of animal science and technology, northwest a & f university. genomic dna was extracted and purified from whole blood following standard procedures and quantified by spectrophotometry and agarose gel electrophoresis. total rna was isolated from flash-frozen tissues. first-strand cdna was synthesized from ng of total rna with the prime script rt reagent kit according to the manufacturer’s instructions. array cgh platform we quantified copy number by hybridizing dna to nimblegen3x720k cgh array, which provided an evenly distributed coverage of ~ oligonucleotide probes. the probes of 50–75 bp in length were designed with similar melting temperatures based on btau_ genome assembly. we chose one pure-blooded angus bull as the reference. dna labelling, hybridization, washing, array scanning, and array imaging were carried out according to the previously described. briefly, pairs of genomic dna were labeled with fluorescent dyes cy or cy, and were co-hybridized on hybridization platform. the arrays were scanned and fluorescent intensity raw data was extracted. the initial data analysis was performed on nimblescan v software with segmnt algorithm. we used an updated version of the previously described method to do cnv calling, ie., determining copy number gains and losses by changes in log signal intensity. the segment, with mean log ratio ≥ | | and at least consecutive probes covered, was defined as a cnv. cnvrs in one group were determined by aggregating overlapped cnvs of all samples. cattle gene annotations were downloaded from the ucsc genome browser, and cattle quantitative trait loci were from the animal qtl database. the genome positions were converted among genome assemblies of btau_, btau_, and btau_. by using the ucsc binary software liftover. we wrote perl scripts to search for gene content and quantitative trait loci inside cnvrs, and determined a positive gene/qtl by > 50% overlap. gene ontology identifiers were retrieved with refgene ids in r package biomart and plotted by the web histogram tool wego. cnvr chromosome plotting, clustering analysis, nonmetric multidimensional scaling, go identifier retrieving, and indicator species analysis were performed by using r packages of ggbio, pvclust, vegan, biomart, and indicspecies, repectively. principal component analysis was performed in stamp v. data access raw array cgh data in this study has been deposited in ncbi geo database under accession number of gse quantitative pcr we performed a qpcr analysis to validate copy number changes detected by array cgh based on the relative comparative cycle threshold method. primers were designed by using beacon designer™. pcr reaction was done in a volume of 20 μl containing 20 ng of genomic dna,  μm of each primer, and sybr premix ex taq™ ii reagents. analysis of resultant crossing thresholds was performed based on the ΔΔct method, and ΔΔct values were determined by comparing test samples and angus reference with btf gene as internal control. finally, the relative copy number for each sample was calculated as 2-ΔΔct. we selected two qpcr-confirmed cnvr and cnvr for further analysis. first, we used the primers to determine cnvr types in individuals against the angus reference. then we selected checked individuals for gene expression analysis of pla2g2d in cnvr and myh in cnvr on the cfx- real-time pcr detection system. two primer pairs were used for expression detection of pla2g2d and myh3: pla2-f, 5′-gacatactggacctgaac-3′; pla2-r, 5′- agccatagtgtgaatagaag-3′; myh3-f, 5′-agtcgtcagttggaggaa-3′; myh3-r, 5′-gctcttctatttgctgggtaa-3′. gapdh gene was used for normalization. the reaction was performed in a volume of 25 μl, containing  μl sybr premix ex taq ii, 1 μl of each primer, 2 μl cdna, and  μl h2o. the relative fold change was calculated using 2-ΔΔct. mean expression levels and standard deviations were obtained by repeating three independent experiments. association analysis between cnvr types and growth traits we evaluated all kinds of factors, and selected three major ones of farm, genotype and breed to build a reduced adjusted linear model: where yijk is trait measurement, μ is overall population mean, fi is farm, gj is genotype effect, bk is breed, and eijk is random error. we used the least-squares means to estimate the association between cnvr types and body measurements in spss software.  cnvrs in cattle groups in taurine, we identified cnvrs covering the region of 47 mb on the placed chromosomes, together with cnvrs on chrunall. all cnvrs correspond to % of the bovine genome, which consist of loss, gain and both events. loss events are approximately 3-fold more than gain ones, while both ones are much longer than the others on average. furthermore, cnvrs are only found in one individual, and cnvrs are shared in two or more ones, among which multiple events have a frequency of ≥ . in yak/buffalo groups, we identified 161/ candidate cnvrs, which consist of 123/ loss, 34/ gain, and 4/ both events. duplications provide additional copies of genes, and this kind of redundancy can allow more flexibilities of gene loss by selective pressure. mutation and selection can result in functional changes introducing a new function or specialization of old functions. thus, gain events might be the first step for extra genetic material during cattle breed formation, and their followed isolated genome cnv evolution and adaption may be a potent evolutionary force for more loss events.table cnvr summary for chinese bulls *percentage of total cnvr length in the genome assembly of btau_. “avg” is for cnvrs per sample. genomic distribution of cnvrs in chinese bulls. cnvrs in bulls are distributed across chromosomes, in which the above are for taurine, while below are the cnvrs for yak and buffalo. refgenes from ucsc genome browser are shown inside the chromosomes in black. on combining three groups, we identified cnvrs totally, % of the bovine genome, among which ones in yak and ones in buffalo are shared by taurine, and about cnvrs are overlapped in all three groups. we then compared shared cnvrs on the placed chromosomes to those in previous studies, and confirmed them all, except for cnvr and cnvr which indicated their reliability. it is notable that cnvr frequencies in different studies are shown diverse, and more shared cnvrs are detected based on cgh and re-sequencing methodologies than that of snp-array-based studies. and cattle breeds, which may have experienced different selection pressures, contribute a lot to cnvr differences as well. it concludes that samples and platform may have the greatest effects on cnv detection.figure venn diagram of cnvrs in three groups. cnvrs are overlapped among taurine, yak, and buffalo in the specific area. cnvrs shared by this study and other studies on cattle cnv *shared cnvrs do not contain the ones on chrunall. we performed an integrated hybridization on the three bovines in one cgh platform, and gained an advantage of parallel comparison, regardless of how different their genomes are. our array probes were designed based on genome assembly btau_. yak genome had been released after our cgh experiment was done, and shown very similar to that of cow. up to date, the complete genome of buffalo is not placed well in ncbi, and only unplaced sequences are ready to download. all our cnvrs in yak and buffalo are from the orthologous regions of cow genome, and our shared cnvrs are supposed to be from the common orthologs of the three bovine genomes. however, their genome positions in yak and buffalo may be different and should be considered cautiously. and the yak- and buffalo-specific regions are not covered by our probes, and out of our scope. it is noted that the bovine cgh array had also been used to scan cnv of goat and sheep. in addition, we designed five probes covering the mitochondrial region. although there is only one cnvr in chrm in three groups, we found a difference between taurine and yak & buffalo. the mitochondrion dna copy number varies referring to energetic metabolism among cell types and fertility between oocytes and sperm. multiple copies of mtdna in the same mitochondrion are directly associated with the amount of atp synthesized. our finding on mtdna copy number difference suggests possible low energetic metabolism in yak and buffalo’s blood, and more studies are necessary to understand the role of mitochondrial copy number in cattle’s traits and performances. we had discovered cnvrs’ distribution preferences across chromosomes. just like the previous results, cattle cnvs are distributed in a non-random way in this study, and their contents vary across chromosomes. the proportion of any known chromosome susceptible to cnvrs ranges from to %, although chrunall shows the strongest enrichment of cnvrs, probably due to highly repetitive sequences in these unplaced contigs. except for chrm, we got strong cnv-enriched chromosomes of × and, which might have been shaped by local chinese bulls, compared with other results. clustered cattle cnvrs selection has also been shown to shape the architecture of segmental duplications during human genome evolution, and studying cnvs’ evolution may help us reveal the genomic alteration and environmental driving impact. the cluster analysis of cnvr in cattle and pig had evidenced that cnvr loci are consistent with the breed divergence and history. so we performed a clustering analysis of cnvrs on all individuals, which revealed remarkable profiles among groups. first, the three groups of taurine, yak, and buffalo are clearly divided. second, individuals in one single breed are easily clustered closely. the clustering results simply showed a phylogeny, while the principal component analysis results showed their detailed relations. the pca plotted samples into three groups. similarly, we investigated the impact of groups on cnvrs structure by using nonmetric multidimensional scaling, which is to visualize the interrelationships among a complex dataset and level of similarity of individuals, and generally grouped samples into taurine, yak, and buffalo. the results supported the hypothesis that genome structure variations, especially cnvs, may be raised by isolated evolutions and shaped by breed formation and adaptation. to highlight potential evolutionary contributions of cnvs to chinese major cattle breeds’ formation and adaptation, we identified cnvrs which are abundant statistically in three groups by using indicator species analysis. compared to shared cnvrs from the orthologs, the biased ones reflect their unique genomic backgrounds, which suggest a potential variance of the three bovine genomes. our parallel comparisons based on one single cgh platform are reliable, which overcome the shortcoming that it is difficult to compare the datasets by different technologies and methods. gene content and quantitative trait loci in cnvrs totally cnvrs encompass genes, which are shown with refgene id and gene name. in order to determine biological functions of copy number variable genes, a gene ontology analysis annotated out of genes in three main go categories: cellular component, molecular function and biological process. as shown in the go map, genes in all categories were mainly involved in eight ones, including cell/cell part, organelle, binding, catalytic activity, metabolic process, cellular process, pigmentation, and biological regulation. this set of copy number variable genes possesses a wide spectrum of molecular functions, and provides a rich resource for hypotheses on their genetic basis of phenotypic variation within and among breeds. many cattle specific genes were also found in our cnvrs, such as c-type lysozymes, bsp30a, interferon tau subfamilies, wc and itln. moreover, eda gene, which has been reported a deletion and responsible for hypotrichosis and dental defects in cattle, and slc4a gene, which is a copy number variable gene and association with osteopetrosis, are both confirmed in our study. we also downloaded cattle qtls from animal qtls database, and searched for potential qtls which reside inside cnvrs on placed chromosomes. there are % of cnvrs overlapped with qtls. the qtls in multiple cnvrs are associated with exterior, health, meat and carcass, milk, production and reproduction. the results are in accordance with chinese breeding history. cnvr confirmation and effects on gene expression to evaluate the accuracy of copy number assignments, quantitative real time-pcr was used as described previously. we selected nine detected cnvr, including loss, gain, and both types, whose frequencies range from % to %. the selected cnvrs were all overlapped with genes and qtls of meat, carcass and production. totally pairs of primers were used, one or two pairs to cover one selected cnvr. the results showed that out of qpcr assays, ones confirm the predictions by array cgh. false-positive identification is common in cnv detection, and there is always to some extent <  of confirming rate. in fact, cnvrs were of complex structure, and qpcr can only target a small portion, which does not reflect their complete characteristics. and the boundaries of cnvr by arrays are indistinct. notably the average size of confirmed cnvrs is  kb, much smaller than those of three unconfirmed ones. certain number of samples are chosen randomly as negative control for the reliability of results. in rats, only 44% genes in cnvrs are differentially expressed. we selected two genes for detailed exploration of expression levels, because both of them might have effects on the performances of cattle. pla2g2d is an innate immunity gene, and thought to play a role in gonadotropin-releasing hormone and mark signaling, which had also been identified inside a cnvr of black angus by re-sequencing method; most recently, it was confirmed that the copy numbers of pla2g2d gene were associated with the index of total merit in holstein bulls. the findings were very important because it was the direct evidence of complex traits of livestock which may be modulated in part by cnvs. and myh is expressed mainly in embryo and muscle, and its mutation caused the freeman-sheldon and sheldon-hall syndrome. in cattle, the snp in myh was also associated with the growth and carcass traits in chinese qinchuan cattle. so, we firstly examined the expression profiles of both genes in qinchuan cattle. the results showed that mrna of pla2g2d was mainly expressed in spleen, intestine, adipose, and lung, while myh mrna was primarily expressed in fetal muscle, and liver, spleen and muscle of calf. secondly, considering both tissue importance in growth traits and mrna expression, we selected samples of adult adipose tissues and ones of calf muscle tissues to analyze cnvr’s effects on mrna of pla2g2d and myh respectively. the results revealed a significant negative correlation between mrna levels of pla2g2d and cnvr. the correlation could be due to position effect of cnvr, and a regulatory sequence of pla2g2d may exactly reside in cnvr. interestingly, the regulatory sequence may be a transcriptional upper repressor and suppressed pla2g2d mrna expression. however, we have not seen any evidence of correlation between myh transcript expression and cnvr it is well known that the break point definition of cnvrs by arrays is equivocal and only % of myh gene is overlapped with cnvr so it is possible that myh or its regulatory motif was not covered by cnvr it is also worth emphasizing that the dosage compensation, lack of regulatory elements in the duplicated copy, differences in the chromatin environment and many other factors might keep mrna levels stable.figure relative mrna expression level of pla2g2d in cnvr in adipose tissues. relative pla2g2d mrna expression levels in adipose tissues of selected qinchuan cattle individuals were analyzed by qpcr, and normalized against that of gapdh. cnv types were determined against the angus reference, and the normal type means the same to the reference. three independent experiments were repeated for reliability. an asterisk denotes a significant difference by t-test. cnvs’ association with growth traits of cattle cnvs may affect phenotype by altering transcriptional level of genes within or adjacent to cnvr and subsequently alter translation levels. the association between the cnvs and production traits of economic interest had been reported. in swine, several copy number variable genes were identified as candidate genes for phenotypes related to carcass length, backfat thickness, abdominal fat weight, length of scapular, intermuscle fat content of logissimus muscle, body weight at 240 day, and fatness. in cattle, the direct evidence of close associations of cnvr# with index of total merit and genetic evaluations for protein production, fat production, and herd life in holstein had been identified. we had demonstrated that the genes inside cnvrs might be expressed differently, and many cnvrs also overlapped with qtls which are associated with cattle performance. so we believed that cnvrs should be potentially associated with cattle body measurements. here we collected chinese cattle samples in qinchuan, jiaxian and nanyang breeds, and evaluated the associations between cnv types and growth traits in equation the results indicate that heart girth and body length are significantly associated with cnv types in cnvr. individuals of loss type have larger heart girth and hucklebone width. the location of cnvr on chromosome exactly fell into qtls with various functions, including qtls and for production, qtls and for meat and carcass in beef cattle. furthermore, pla2g2d gene was overlapped with cnvr cattle of loss type in cnvr with higher pla2g2d expression maybe finally gain more fat deposition. in addition, association between cnv of pla2g2d gene and index of total merit had been reported in holstein.table association analysis of cnvr with body measurements *lsm ± se, “lsm” for least squares mean, “se” for standard error. “n” indicates sample number. a, bmeans with different superscripts were significantly different. on the other hand, we also found that cnvr is significantly associated with heart girth, probably due to the fact that cnvr was overlapped with the qtls for production and meat and carcass in beef cattle. regardless of one single gene’s contributions, we had a direct look at the effects of selected cnvrs on cattle body performances, which might be caused by a group of genes. it took gene population effects into consideration, rather than focusing on only one single trait-related gene. the association between cnvs and traits is inspiring, but larger population may be needed to validate it.  we have performed a comprehensive genomic analysis of cnvs based on cgh arrays in chinese cattle, and a detailed functional investigation for cnvrs’ effects on both gene expression and cattle body measurements. we identified cnvrs in b. taurus, which covered % of the bovine genome, together with and cnvrs in b. grunnies and b. bubalis. furthermore, we confirmed that cnvr had significantly negative effects on both pla2g2d gene expression and cattle body measurements, while cnvr showed a significant negative association with heart girth. our results generated a valuable genome-wide variation resource for chinese cattle genomic researches, and provided a novel insight into understanding the association between animal complex traits and cnvrs during their adapting to local geographical environment and domesticated needs from human society. electronic supplementary material additional file 1: table s: the breeds or populations of chinese cattle used in the cgh arrays. table s. the breeds or populations of chinese cattle used in other experiments. table s primer sequences and results for qpcr. table s cnvrs in chinese bulls. table s shared cnvrs by this study and other studies. table s cnvr in chrm in chinese bulls. table s cnv types of individuals on cnvr and cnvr in three chinese breeds. table s association analysis of cnvr with body measurements. additional file 2: figure s1: clustering results of cnvrs. figure s pca and nmds of cnvrs on chinese bulls. figure s gene ontology annotations for genes covered by cnvrs. figure s expression pattern analysis of pla2g2d gene and myh gene. abbreviations cnvcopy number variations cnvrcopy number variable region cghcomparative genomic hybridization gogene ontology myh3myosin- b. taurus myosin, heavy chain 3 isaindicator species analysis pla2g2db. taurus calcium-dependent phospholipase a2 nmdsnonmetric multidimensional scaling pcaprincipal component analysis qpcrquantitative real time pcr qtlsquantitative trait loci snpsingle nucleotide polymorphism. competing interests the authors declared that they have no competing interests. authors’ contributions lzz and hc conceived and designed the experiments. lzz, mjy and yx performed the experiments. lzz and sgj analyzed the data. czl, xyl, clz, jjs, yzh, yz, cjl, and xz contributed reagents/materials/analysis tools. lzz and sgj wrote and revised the manuscript. all authors read and approved the final manuscript. 
totally, we discovered integrated cnvrs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. finally, we confirmed cnvrs in chosen ones by using quantitative pcr, and further demonstrated that cnvr had significantly negative effects on expression of pla2g2d gene, and both cnvr and cnvr were associated with body measurements in chinese cattle, suggesting their key effects on gene expression and cattle traits. this study provides a highly valuable resource for chinese cattle’s evolution and breeding researches. we identified copy number variable regions, covering % of the bovine genome, in taurine, together with ones in yaks and ones in buffaloes. the results advanced our understanding of cnv as an important genomic structural variation in taurine, yak and buffalo. copy number variations are a main source of genomic structural variations underlying animal evolution and production traits. furthermore, we confirmed approximately % and % cnvrs span cattle genes and quantitative trait loci, respectively. here, with one pure-blooded angus bull as reference, we describe a genome-wide analysis of cnvs based on comparative genomic hybridization arrays in chinese domesticated bulls and examined their effects on gene expression and cattle growth traits. copy number variationsbos taurusbos grunniensbubalus bubalisgene expressionissue-copyright-statement© the author 2014
 copy number variations are a main source of genomic structural variations underlying animal evolution and production traits. here, with one pure-blooded angus bull as reference, we describe a genome-wide analysis of cnvs based on comparative genomic hybridization arrays in chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.  we identified copy number variable regions, covering % of the bovine genome, in taurine, together with ones in yaks and ones in buffaloes. totally, we discovered integrated cnvrs, with more “loss” events than both “gain” and “both” ones, and clearly clustered them into three cattle groups. interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion dna copy number. furthermore, we confirmed approximately % and % cnvrs span cattle genes and quantitative trait loci, respectively. finally, we confirmed cnvrs in chosen ones by using quantitative pcr, and further demonstrated that cnvr had significantly negative effects on expression of pla2g2d gene, and both cnvr and cnvr were associated with body measurements in chinese cattle, suggesting their key effects on gene expression and cattle traits.  the results advanced our understanding of cnv as an important genomic structural variation in taurine, yak and buffalo. this study provides a highly valuable resource for chinese cattle’s evolution and breeding researches. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords copy number variationsbos taurusbos grunniensbubalus bubalisgene expressionissue-copyright-statement© the author 2014 