BACKGROUND
a vast number of eggs are produced annually for human consumption worldwide.
avian calcified eggshell as a unique bioceramic material can provide protection to egg contents from physical damage and microorganic invasion  <cit> .
changes in eggshell properties are directly related to increasing risk of foodborne disease for the consumer  <cit> .
low eggshell quality will also lead to more cracked eggs in the automatic sorting and packing process in the modern egg industrial production  <cit> .
it has been estimated that improving the mean eggshell strength by one newton will lead to  <dig>  % less broken eggs per hen per laying cycle  <cit> .
on the other hand, eggshell is biologically significant for bird embryo developments by allowing gas exchange between the embryo and the environment and supplying calcium for the embryo bone deposition  <cit> .
hatching eggs with thin eggshell have high embryonic mortality, owing to more loss of water vapour during the incubation  <cit> .
furthermore, eggshell are subject to quality loss with the aging process of laying hens  <cit> , which hinders the developmental trend to prolong the laying cycles of egg-type chicken in the future.
therefore, understanding the genetic control for dynamic eggshell quality with aging process is of great economic and biological importance.
in recent years, the genomic  <cit> , transcriptomic , proteomic  and structural analyses  of eggshell have been carried out to provide new insights into better understanding on the eggshell mineralization and its ultrastructure or micostructure that contribute to eggshell quality.
identifying the quantitative trait loci  that relate to eggshell quality is one of the most powerful strategies to illustrate the genetic mechanism underlying eggshell quality.
up to date, a total of  <dig> qtls related to eggshell quality have been collected in the animalqtldb .
however, most previously reported qtls were identified by low-density linkage analysis with limited markers of microsatellites , which restricted the confidence intervals and mapping accuracy  <cit> .
recently, genome-wide association study  has been utilized to identify the associations between genomic loci and phenotypes with relatively high-density snp arrays in chicken  <cit> .
for instance, liu et al.
<cit>  conducted the first gwa analysis with relatively high-density sna array  to refine the associations with egg production and egg quality traits in chicken.
with the rapid advance in next-generation sequencing technologies, large amounts of snps in chicken have been discovered  <cit> .
the development of 600 k affymetrix chicken snp array allows more efficient screening of the causal loci and genes in relevance to target traits.
it is notable that many complex traits are dynamic varied with the aging process of animals  <cit> .
however, previous studies utilized the phenotypes from limited age points.
animal phenotypes at different age should be utilized in gwa analysis to refine the associations with age-dependent complex traits and increase the statistical power.
growing evidences had been provided that this type of “longitudinal design” could efficiently identify the time-dependent or consistent loci for complex traits  <cit> .
in the current study, we conducted gwa analysis on the dynamic eggshell quality traits at  <dig> time points from onset of laying to  <dig> weeks old utilizing a 600 k high-density snp arrays in an f <dig> chicken population.
we aimed to explore the associated genomic loci and genes that contribute to the phenotypic variability and dynamic change in eggshell quality traits, and to provide potential breeding tools for the improvement of eggshell quality.
RESULTS
phenotype description and genetic parameters
means and standard deviations for eggshell quality traits, including esw, est and ess, at  <dig> time points from onset of laying to  <dig> weeks of age are presented in table  <dig> and additional file 1: figure s <dig>  these three eggshell quality traits displayed a smooth curve with hen age except  <dig> weeks-old, at which esw, est and ess showed a abrupt decrease  and the results of gwa analysis in this week were also unusual .
we speculated that this might be caused by the feed or environment changes in this period leading to eggshell quality decrease.
for this reason, phenotypes collected at  <dig> weeks-old were not included in the subsequent multivariate gwa analysis.
the additive genetic variations of esw, est and ess at different age were estimated from all eligible markers using gcta.
esw at all ages exhibited moderate snp-based heritability estimates , and the highest snp-based heritability estimate was found in esw <dig> .
however, the estimates for est  and ess  were relatively low.
moreover, bivariate gcta analyses indicated that esw at different age were highly and positively correlated, specially at neighboring time points .table  <dig> descriptive statistics of eggshell traits in the f
<dig> population
n number of samples, esw eggshell weight, est eggshell thickness, ess eggshell strength, mean arithmetic mean, sd standard deviation, afe age of first egg
traitsa
diagonal: heritability estimates.
lower triangle: phenotypic correlations.
upper triangle: genetic correlations.
standard errors of the estimates are in parentheses
a
fesw eggshell weight of first egg, esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig> eggshell weights of  <dig> , <dig> , <dig> , <dig> , <dig>  weeks of age
identification of candidate loci by gwas
eggshell quality traits were collected at  <dig> time points from onset of laying to  <dig> weeks.
at each time point, three separate genome-wide association tests were conducted for each eggshell quality trait with univariate method.
a total of  <dig>   <dig> and  <dig> genome-wide significant associations were identified with esw, est and ess, respectively .
almost all the significant locus were in a region spanning from  <dig>  to  <dig>  m of chromosome  <dig>  .
out of these loci in this region,  <dig> snps were observed in association with all these three eggshell quality traits at genome-wide significance level .
it was notable that most of the snps in this region possess a maf more than  <dig> .
in addition,  <dig> loci on chromosome  <dig>  were significantly associated with esw, but only for young hens from age of first egg  to  <dig> week old.
the global view of the putative p-values for all snps affecting eggshell quality traits at 44weeks is given in fig.
<dig>  and the manhattan and quantile-quantile  plots for the remaining time points in additional file 1: figure s <dig> table  <dig> number and distribution of significant snps for eggshell traits
afe age of first egg, esw eggshell weight, est eggshell thickness, ess eggshell thickness
amulti-analysis means using multivariate model for gwa analysis.
for the analysis of esw and ess, time points of  <dig> weeks, and 40– <dig> weeks were used; for est, time points of 40– <dig> weeks were used
fig.
<dig> venn diagram of significant snps associated with three eggshell quality traits by univariate  and multivariate  model.
eggshell weight, eggshell thickness and eggshell strength were abbreviated as esw, est and ess respectively
fig.
<dig> manhattan plot  and quantile-quantile plot  of the observed p-values for esw , est  and ess  at  <dig> weeks of age .
the manhattan plot indicates -log <dig>  for genome-wide snps  plotted against their respective positions on each chromosome , and the horizontal green and black lines depict the genome-wide suggestive  and significant  threshold, respectively.
for quantile-quantile plot, the x-axis shows the expected -log10-transformed p-values, and the y-axis represents the observed -log10-transformed p-values.
the genomic inflation factors  are shown on the top left in the qq plot.
green points represent the genome-wide significant associations
to enhance the statistical power, a joint gwa analysis of multi time points was performed by fitting these snps into a multivariate model.
the time points that had significant snps were included in this model except for  <dig> weeks owing to the abnormal phenotype in this week.
the  <dig> significant snps related to eggshell quality identified by univariate method in gga <dig> were further analyzed in this multivariate model.
consequently, a total of  <dig>   <dig> and  <dig> significant hits on gga <dig> from  <dig>  to  <dig>  m presented consistent and compelling associations with longitudinal eggshell quality traits , and  <dig> snps out of these locus had pervasive effect on esw, est and ess .
linkage disequilibrium  and conditional analysis
the linkage disequilibrium  analysis were then carried out and the results showed the uncovered snps in gga <dig> were in extremely strong linkage disequilibrium status, especially for the ld block from  <dig>  to  <dig>  mb that include half  of the total loci .
to detect the independent acting locus in this region, we then carried out stepwise conditional gwa analysis to prioritize separately associated snps.
at locus rs <dig>  a missense mutation in association with all three eggshell traits was then fitted into the multivariate model using its genotype as covariate to explore the change of the associations with other loci.
as shown in fig.
<dig>  if the genotype of rs <dig> was used as covariate in multivariate analysis, the significance levels of all other loci were substantially attenuated below genome-wide significant level.
regional plots and conditional analysis in multivariate model for the traits of eggshell thickness  and eggshell strength  were shown in additional file 1: figure s <dig> fig.
<dig> regional plot and conditional analysis in multivariate model for esw.
plot a: up to  <dig> significant snps  obtained by univariate model were re-analyzed for their association with esw by multivariate model .
the -log <dig>  of snps  are presented according to their chromosomal positions .
totally  <dig> snps  reached genome-wide significance level .
plot b: the genotype of rs <dig> was put into the multivariate model as covariances for conditional analysis.
after conditioning on rs <dig>  the significant snps in plot a  were all substantially attenuated below genome-wide significant level in plot b. plot c: regional plot shows extremely strong linkage disequilibrium status exist in this  <dig>  mb region, especially for the ld block from  <dig>  to  <dig>  mb
estimation of contribution to phenotypic variation
the phenotypic variance explained by loci or genomic region were estimated by a tool of gcta for three eggshell traits.
all together,  <dig> significant loci from  <dig>  to  <dig>  mb in gga <dig> could account for  <dig>  ~  <dig>  % of the phenotypic variance for esw from  <dig> to  <dig> weeks of age,  <dig>  ~  <dig>  % and  <dig>  ~  <dig>  % for est and ess from  <dig> to  <dig> weeks of age.
for single locus, five missense mutations out of these  <dig> loci were estimated for their cpvs .
among these five loci, two snps presented high cpvs for eggshell traits.
one loci, rs <dig>  could independently explain  <dig> – <dig>  %,  <dig> – <dig>  % and  <dig> – <dig>  % of the phenotypic variance for esw, est and ess from  <dig> to  <dig> weeks of age respectively.
notably, the effect alleles at rs <dig> were associated with esw at each time point except the first egg.
the other one, rs <dig>  could account for  <dig> – <dig>  %,  <dig> – <dig>  % and  <dig> – <dig>  % of the phenotypic variance respectively.table  <dig> contributions of five missense mutations and genomic regions to eggshell quality traits at different wk of age
rs312347405
rs14837998
rs316793137
rs14840004
rs316607577
region 1
ea/aaa
ea effect allele , aa alternative allele , maf minor allele frequency, cpv contribution to phenotypic variance , esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig>  esw <dig> eggshell weights of  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> weeks of age
aestimated allelic substitution effect per copy of the effect allele ; se standard error of the beta bbeta means the effect size of minor alleles.
positive/negative effect size value means that the substitution of major allele to minor allele can lead to heavier/lighter yolk or ovary weight
the phenotypic differences among  <dig> genotypes at these two loci are displayed in fig.
<dig>  there was no difference in eggshell quality traits among different genotypes for the first egg.
however, along with the aging process, profound differences in eggshell quality appeared among three genotypes.
eggshell of each genotype exerted different rate of quality decay with aging process, i.e., the homozygotes of advantageously effective alleles possessed the best eggshell quality and could partly counteract the negative effect of aging process.
however, the change trends of eggshell traits among three genotypes were concordant along with age.fig.
<dig> phenotypic differences contributed by loci of rs <dig> and rs <dig> on genes of pik3c2g and itpr <dig>  the left three plots describe the phenotypes of esw, est and ess among three genotypes at rs <dig>  the right three plots describe the phenotypes of esw, est and ess among three genotypes at rs <dig>  red square, black circle and blue triangle denote minor-allele homozygotes, heteorozygotes and major-allele homozygotes, respectively.
number of samples for each genotype is indicated in the top or bottom left
promising genes related to eggshell quality
utilizing gene annotation of the causal locus allowed us to screen the putative genes relating to eggshell quality.
for snps-traits association analysis, the missense mutations on exons were more meaningful than the snps located on introns or intergenic regions.
we totally found five missense loci at gga <dig> and one at gga <dig> using univariate genome-wide screens.
they located on  <dig> independent genes including phosphatidylinositol–4- phosphate 3-kinase, catalytic subunit type  <dig> gamma , inositol  <dig> ,5-trisphosphate receptor type  <dig> , recq helicase-like , atp-binding cassette sub-family c member  <dig>  and cancer susceptibility candidate  <dig>  on gga <dig> and non-smc condensin i complex subunit g  on gga <dig>  nonetheless, only two snps, rs <dig> and rs <dig>  located on pik3c2g and itpr <dig>  remained significantly associated with eggshell quality traits after multivariate gwa analysis.
furthermore, as illustrated above, these two snps could explain more phenotypic variance than others.
consequently, we first considered pik3c2g and itpr <dig> as the primary candidate genes associated with eggshell quality.
the missense mutation in gga <dig> that located on ncapg was posterior putative locus for its roles in affecting early eggshell weight.
in addition to missense mutations on exons, the mutations on 3′ or 5′ utrs were also of biological significance for genetic variation underlying complex traits.
we aggregately discovered  <dig> mutations that located on 3′ or 5′ utrs corresponding to  <dig> independent genes .
according to functional annotation and database research, however, most of these genes had no direct relevance to eggshell calcification or calcium metabolism, expect for rs <dig> which located on the 3′utr of itpr <dig> gene.
the detailed message of promising loci and the corresponding candidate genes are listed in table  <dig> table  <dig> putative genes associated with eggshell quality
gene symbola
chr chromosome, aa amino acid, sift sorting intolerant from tolerant
aidentification of the gene according to ensembl genes database 76
bsift is a program that predicts whether an amino acid substitution affects protein function.
small values means deleterious amino acid change
discussion
it is known that eggshells are subject to quality loss along with the age process  <cit> .
in egg-type chicken production, there exists a trend to prolong the laying cycles  <cit> , whereas the decline of eggshell quality for old hens proposes a substantial challenge for this development pattern.
in the present study, we elucidated genomic architecture underlying the age-dependent dynamic eggshell quality, which has biological and practical significance.
the qtl number detected for eggshell quality was limited in previous studies.
the low to medium heritability of the eggshell trait revealed by our results and other study  <cit>  suggested the necessary use of large population for causal mutant identification.
our f <dig> population consisting of  <dig> hens is the largest population used for eggshell quality gwa analysis so far, and therefore the novel genomic region and loci revealed by the current study should be accurate and reliable.
our gwa analysis for dynamic eggshell quality traits identified thousands of significant associations, that were much more than those screened by previous gwa studies in chicken  <cit> .
these significant mutations were almost in a same region with an extremely strong linkage disequilibrium status, which may result from the insufficient recombination events in the f <dig> segregation population  <cit> .
lots of qtl regions affecting esw, est and ess were discovered by previous linkage studies  <cit>  and mainly distributed on gga 1– <dig> and gga <dig>  the promising genomic region from  <dig>  to  <dig>  mb identified in the current study had no overlap with the previously reported regions in gga  <dig>  mainly on  <dig> – <dig>  mb,  <dig> – <dig>  mb,  <dig> – <dig>  mb and  <dig> – <dig>  mb .
the  <dig> significant snps in this region were distributed on  <dig> independent genes.
we tried to perform gene ontology   <cit>  and pathway analysis  <cit>  on these  <dig> genes, whereas no significant go terms or pathways were enriched.
this suggested there were not a series of functionally similar genes in this region to influence eggshell quality together.
most snps might passively display significant associations for their linkages to a sort of real causal mutants on one or a few genes.
hence, the phenotypic variance explained by all loci in this region was only slightly higher than single locus.
it was notable that more associations were discovered for old hens  than young hens , suggesting certain genetic variants were age-dependent.
this was partially consistent with the other gwa studies, indicating no significant associations with both early and late shell quality  <cit> .
however, our results also proved the existence of common genetic variants consistently influencing eggshell quality from  <dig> to  <dig> weeks.
six significant missense mutations in this genomic region were considered as the most important putative variants.
after multivariate gwa analysis, only two loci, rs <dig> and rs <dig> on pik3c2g and itpr <dig> in gga <dig> remained significantly associated with dynamic eggshell quality traits.
the loci of rs <dig> on pik3c2g could explain the most phenotypic variance of eggshell quality traits among the five missense mutations.
hens with homozygotes of advantageously effective alleles  could produce eggs with excellent ess, which decreased relatively within a narrow range along with the aging process.
pik3c2g encoded proteins belonging to the phosphoinositide 3-kinase  family, containing a lipid kinase catalytic domain as well as a c-terminal c <dig> domain  <cit> , which acted as calcium-dependent phospholipid binding motifs  <cit> .
previous proteomic screens revealed that a high proportion of lipid-binding proteins abundantly existed in eggshell matrix such as extracellular fatty acid-binding protein , prosaposin and apolipoprotein d  <cit> .
considering the low abundance of lipids in eggshell, the existence of high abundance of lipid-binding proteins in eggshell matrix was perplexed  <cit> .
furthermore, one association analysis revealed another lipid-related gene, low-density lipoprotein receptor-related protein  <dig>  gene, as a new member of eggshell matrix protein, was significantly associated with eggshell traits  <cit> .
now in the current study pik3c2g possessing the c <dig> domain acting as lipid binding motif was also implicated in eggshell property.
the role of lipid binding proteins in eggshell or during eggshell formation should be re-recognized.
pik3c2g possessing the c <dig> domain could mediate translocation of proteins to lipid membranes, and also regulate protein-protein interactions in human and mammals  <cit> .
the interoperable matrix proteins and calcite together formed the bioceramic eggshell  <cit> .
it could be hypothesized that lipid binding proteins might act as carrier or modifier of matrix precursors, which mediate eggshell calcification process or act as frame proteins deposited into eggshell structure.
consequently, pik3c2g was considered as candidate gene for eggshell quality.
the gene of inositol  <dig> ,5-trisphosphate receptor type  <dig>  harboring the loci of rs <dig>  was the positional and functional candidate gene for eggshell quality revealed by the current gwa analysis.
the mutation of rs <dig> was a nonconservative substitution of serine by glycine , with the glycine-encoding allele being associated with stronger eggshell.
a gene could be inactivated by a mutation either in a control site or in a coding region  <cit> .
so rs <dig> on 3′urt of itpr <dig> was also the susceptibility locus.
itpr <dig> was known for its mediation in endoplasmic reticulum  calcium release and inositol  <dig> ,5-trisphosphate  could mobilize ca2+ from intracellular calcium stores to many types of cells  <cit> .
the existence of itpr <dig> in uterine epithelial cell of chicken had been verified  <cit> , furthermore, the expression of itpr <dig> in uterus during eggshell calcification was significantly higher than that in magnum and duodenum which also possess active calcium metabolism  <cit> .
this provided the evidences that itpr <dig> play roles in the regulation of intra-cellular ca2+ transportation in uterus and contributed to the process of eggshell calcification.
recently, a genome-wide association study in humans identified itpr <dig> as a susceptibility gene for kashin-beck disease which is a chronic osteochondropathy  <cit> , mainly characterized by cartilage degeneration, cartilage matrix degradation, chondrocyte necrosis and apoptosis  <cit> .
this uncovered association with bone disease indicated that it might play a role in general mineralization processes.
birds process medullary bone, a nonstructural type of woven bone, to act as a reservoir for the minerals required for shell calcification  <cit> .
our previous comparative proteomic analysis for uterine fluid and eggshell matrix proteins also identified that many bone-development related proteins were up-presented in strong eggshell group  <cit> .
up to date, there only existed one paper referred to the association of ion transporter genes with eggshell quality.
they found one snp on a sodium channel gene  had effects on eggshell strength  <cit> , however, the phenotypic variance of ess it could explained was relatively low  compared to that of itpr <dig>  in the current study.
all the above evidences suggested itpr <dig> should be a crucial and promising candidate gene relating to eggshell calcification as well as eggshell mechanical property.
another missense mutation  located on non-smc condensin i complex subunit g  gene in gga <dig> was discovered in association with eggshell weight for young hens in our gwa analysis.
many other studies reported multiple genomic regions containing this gene were identified to be associated with body weight  <cit>  and egg weight  <cit>  in chickens and many other growth traits in livestocks  <cit> .
the current study suggested that ncapg also involved in influencing eggshell weight.
larger eggs generally owned heavier eggshell.
hence we could not exclude the potential cause that the significant association of ncapg with esw was just due to its relevance to egg weight.
nevertheless, we still considered ncapg as a candidate gene for its consistent association with eggshell weight from  <dig> to  <dig> week.
previous studies hypothesized that eggshell organic matrix as a complicated mixture of proteins might play a regulatory role in assembly of the calcite zone with calcium carbonate to ultimately determine its strength, and many evidences had been proposed to support this  <cit> , such as the different matrix protein concentrations in strong and weak eggshells  <cit> .
furthermore, many works conducted to investigate the associations between polymorphisms in genes encoding eggshell matrix proteins and eggshell quality traits  <cit> .
takahashi et al.
<cit>  conducted association analysis between ovocalyxin- <dig> gene haplotypes and eggshell quality traits in an f <dig> intercross and dunn et al.
<cit>  carried out association analysis between polymorphisms in  <dig> eggshell organic matrix genes and eggshell quality measurements in two lines.
these two studies found some significant associations, however, the loci could only explain limited phenotypic variances.
our current study discovered no association between snps on eggshell matrix genes and eggshell quality, suggesting that the influences of eggshell matrix genes on eggshell quality were not caused by their nucleotide polymorphisms.
CONCLUSIONS
the dynamic eggshell quality traits at  <dig> time points from onset of laying to  <dig> weeks were collected and used for genome-wide association analysis with a 600 k high-density snp array.
according to univariate and multivariate gwa analysis, we discovered a genomic region spanning from  <dig>  to  <dig>  m in gga <dig> significantly associated with eggshell quality.
ld and conditional analysis suggested this region were in extremely strong linkage disequilibrium status, especially for the ld block from  <dig>  to  <dig>  mb that almost include half  of the total significant loci.
ultimately, three genes, pik3c2g, itpr <dig> and ncapg identified from three missense mutations were considered as promising candidate genes that implicated in dynamic eggshell quality.
the homozygotes of advantageously effective alleles on pik3c2g and itpr <dig> possessed the best eggshell quality and could partly counteract the negative effect of aging process.
these promising loci and genes could be helpful to engineer practical breeding programs for the improvement of eggshell quality for old hens to meet the need of prolonging the laying cycle.
the new findings also have greatly advanced our understanding of the genetic basis underlying eggshell quality.

 a vast number of eggs are produced annually for human consumption worldwide. avian calcified eggshell as a unique bioceramic material can provide protection to egg contents from physical damage and microorganic invasion. changes in eggshell properties are directly related to increasing risk of foodborne disease for the consumer. low eggshell quality will also lead to more cracked eggs in the automatic sorting and packing process in the modern egg industrial production. it has been estimated that improving the mean eggshell strength by one newton will lead to  % less broken eggs per hen per laying cycle. on the other hand, eggshell is biologically significant for bird embryo developments by allowing gas exchange between the embryo and the environment and supplying calcium for the embryo bone deposition. hatching eggs with thin eggshell have high embryonic mortality, owing to more loss of water vapour during the incubation. furthermore, eggshell are subject to quality loss with the aging process of laying hens, which hinders the developmental trend to prolong the laying cycles of egg-type chicken in the future. therefore, understanding the genetic control for dynamic eggshell quality with aging process is of great economic and biological importance. in recent years, the genomic, transcriptomic, proteomic and structural analyses of eggshell have been carried out to provide new insights into better understanding on the eggshell mineralization and its ultrastructure or micostructure that contribute to eggshell quality. identifying the quantitative trait loci that relate to eggshell quality is one of the most powerful strategies to illustrate the genetic mechanism underlying eggshell quality. up to date, a total of qtls related to eggshell quality have been collected in the animalqtldb. however, most previously reported qtls were identified by low-density linkage analysis with limited markers of microsatellites, which restricted the confidence intervals and mapping accuracy. recently, genome-wide association study has been utilized to identify the associations between genomic loci and phenotypes with relatively high-density snp arrays in chicken. for instance, liu et al. conducted the first gwa analysis with relatively high-density sna array to refine the associations with egg production and egg quality traits in chicken. with the rapid advance in next-generation sequencing technologies, large amounts of snps in chicken have been discovered. the development of 600 k affymetrix chicken snp array allows more efficient screening of the causal loci and genes in relevance to target traits. it is notable that many complex traits are dynamic varied with the aging process of animals. however, previous studies utilized the phenotypes from limited age points. animal phenotypes at different age should be utilized in gwa analysis to refine the associations with age-dependent complex traits and increase the statistical power. growing evidences had been provided that this type of “longitudinal design” could efficiently identify the time-dependent or consistent loci for complex traits. in the current study, we conducted gwa analysis on the dynamic eggshell quality traits at time points from onset of laying to weeks old utilizing a 600 k high-density snp arrays in an f chicken population. we aimed to explore the associated genomic loci and genes that contribute to the phenotypic variability and dynamic change in eggshell quality traits, and to provide potential breeding tools for the improvement of eggshell quality.  phenotype description and genetic parameters means and standard deviations for eggshell quality traits, including esw, est and ess, at time points from onset of laying to weeks of age are presented in table  and additional file 1: figure s these three eggshell quality traits displayed a smooth curve with hen age except weeks-old, at which esw, est and ess showed a abrupt decrease and the results of gwa analysis in this week were also unusual. we speculated that this might be caused by the feed or environment changes in this period leading to eggshell quality decrease. for this reason, phenotypes collected at weeks-old were not included in the subsequent multivariate gwa analysis. the additive genetic variations of esw, est and ess at different age were estimated from all eligible markers using gcta. esw at all ages exhibited moderate snp-based heritability estimates, and the highest snp-based heritability estimate was found in esw. however, the estimates for est and ess were relatively low. moreover, bivariate gcta analyses indicated that esw at different age were highly and positively correlated, specially at neighboring time points.table descriptive statistics of eggshell traits in the f population n number of samples, esw eggshell weight, est eggshell thickness, ess eggshell strength, mean arithmetic mean, sd standard deviation, afe age of first egg traitsa diagonal: heritability estimates. lower triangle: phenotypic correlations. upper triangle: genetic correlations. standard errors of the estimates are in parentheses a fesw eggshell weight of first egg, esw esw esw esw esw esw esw esw esw esw eggshell weights of,,,, weeks of age identification of candidate loci by gwas eggshell quality traits were collected at time points from onset of laying to weeks. at each time point, three separate genome-wide association tests were conducted for each eggshell quality trait with univariate method. a total of and genome-wide significant associations were identified with esw, est and ess, respectively. almost all the significant locus were in a region spanning from to  m of chromosome. out of these loci in this region, snps were observed in association with all these three eggshell quality traits at genome-wide significance level. it was notable that most of the snps in this region possess a maf more than. in addition, loci on chromosome were significantly associated with esw, but only for young hens from age of first egg to week old. the global view of the putative p-values for all snps affecting eggshell quality traits at 44weeks is given in fig.  and the manhattan and quantile-quantile plots for the remaining time points in additional file 1: figure s table number and distribution of significant snps for eggshell traits afe age of first egg, esw eggshell weight, est eggshell thickness, ess eggshell thickness amulti-analysis means using multivariate model for gwa analysis. for the analysis of esw and ess, time points of weeks, and 40– weeks were used; for est, time points of 40– weeks were used fig. venn diagram of significant snps associated with three eggshell quality traits by univariate and multivariate model. eggshell weight, eggshell thickness and eggshell strength were abbreviated as esw, est and ess respectively fig. manhattan plot and quantile-quantile plot of the observed p-values for esw, est and ess at weeks of age. the manhattan plot indicates -log for genome-wide snps plotted against their respective positions on each chromosome, and the horizontal green and black lines depict the genome-wide suggestive and significant threshold, respectively. for quantile-quantile plot, the x-axis shows the expected -log10-transformed p-values, and the y-axis represents the observed -log10-transformed p-values. the genomic inflation factors are shown on the top left in the qq plot. green points represent the genome-wide significant associations to enhance the statistical power, a joint gwa analysis of multi time points was performed by fitting these snps into a multivariate model. the time points that had significant snps were included in this model except for weeks owing to the abnormal phenotype in this week. the significant snps related to eggshell quality identified by univariate method in gga were further analyzed in this multivariate model. consequently, a total of and significant hits on gga from to  m presented consistent and compelling associations with longitudinal eggshell quality traits, and snps out of these locus had pervasive effect on esw, est and ess. linkage disequilibrium and conditional analysis the linkage disequilibrium analysis were then carried out and the results showed the uncovered snps in gga were in extremely strong linkage disequilibrium status, especially for the ld block from to  mb that include half of the total loci. to detect the independent acting locus in this region, we then carried out stepwise conditional gwa analysis to prioritize separately associated snps. at locus rs a missense mutation in association with all three eggshell traits was then fitted into the multivariate model using its genotype as covariate to explore the change of the associations with other loci. as shown in fig.  if the genotype of rs was used as covariate in multivariate analysis, the significance levels of all other loci were substantially attenuated below genome-wide significant level. regional plots and conditional analysis in multivariate model for the traits of eggshell thickness and eggshell strength were shown in additional file 1: figure s fig. regional plot and conditional analysis in multivariate model for esw. plot a: up to significant snps obtained by univariate model were re-analyzed for their association with esw by multivariate model. the -log of snps are presented according to their chromosomal positions. totally snps reached genome-wide significance level. plot b: the genotype of rs was put into the multivariate model as covariances for conditional analysis. after conditioning on rs the significant snps in plot a were all substantially attenuated below genome-wide significant level in plot b. plot c: regional plot shows extremely strong linkage disequilibrium status exist in this  mb region, especially for the ld block from to  mb estimation of contribution to phenotypic variation the phenotypic variance explained by loci or genomic region were estimated by a tool of gcta for three eggshell traits. all together, significant loci from to  mb in gga could account for  ~   % of the phenotypic variance for esw from to weeks of age,  ~   % and  ~   % for est and ess from to weeks of age. for single locus, five missense mutations out of these loci were estimated for their cpvs. among these five loci, two snps presented high cpvs for eggshell traits. one loci, rs could independently explain –  %, –  % and –  % of the phenotypic variance for esw, est and ess from to weeks of age respectively. notably, the effect alleles at rs were associated with esw at each time point except the first egg. the other one, rs could account for –  %, –  % and –  % of the phenotypic variance respectively.table contributions of five missense mutations and genomic regions to eggshell quality traits at different wk of age rs312347405 rs14837998 rs316793137 rs14840004 rs316607577 region 1 ea/aaa ea effect allele, aa alternative allele, maf minor allele frequency, cpv contribution to phenotypic variance, esw esw esw esw esw esw esw eggshell weights of weeks of age aestimated allelic substitution effect per copy of the effect allele; se standard error of the beta bbeta means the effect size of minor alleles. positive/negative effect size value means that the substitution of major allele to minor allele can lead to heavier/lighter yolk or ovary weight the phenotypic differences among genotypes at these two loci are displayed in fig.  there was no difference in eggshell quality traits among different genotypes for the first egg. however, along with the aging process, profound differences in eggshell quality appeared among three genotypes. eggshell of each genotype exerted different rate of quality decay with aging process, i.e., the homozygotes of advantageously effective alleles possessed the best eggshell quality and could partly counteract the negative effect of aging process. however, the change trends of eggshell traits among three genotypes were concordant along with age.fig. phenotypic differences contributed by loci of rs and rs on genes of pik3c2g and itpr the left three plots describe the phenotypes of esw, est and ess among three genotypes at rs the right three plots describe the phenotypes of esw, est and ess among three genotypes at rs red square, black circle and blue triangle denote minor-allele homozygotes, heteorozygotes and major-allele homozygotes, respectively. number of samples for each genotype is indicated in the top or bottom left promising genes related to eggshell quality utilizing gene annotation of the causal locus allowed us to screen the putative genes relating to eggshell quality. for snps-traits association analysis, the missense mutations on exons were more meaningful than the snps located on introns or intergenic regions. we totally found five missense loci at gga and one at gga using univariate genome-wide screens. they located on independent genes including phosphatidylinositol–4- phosphate 3-kinase, catalytic subunit type gamma, inositol,5-trisphosphate receptor type, recq helicase-like, atp-binding cassette sub-family c member and cancer susceptibility candidate on gga and non-smc condensin i complex subunit g on gga nonetheless, only two snps, rs and rs located on pik3c2g and itpr remained significantly associated with eggshell quality traits after multivariate gwa analysis. furthermore, as illustrated above, these two snps could explain more phenotypic variance than others. consequently, we first considered pik3c2g and itpr as the primary candidate genes associated with eggshell quality. the missense mutation in gga that located on ncapg was posterior putative locus for its roles in affecting early eggshell weight. in addition to missense mutations on exons, the mutations on 3′ or 5′ utrs were also of biological significance for genetic variation underlying complex traits. we aggregately discovered mutations that located on 3′ or 5′ utrs corresponding to independent genes. according to functional annotation and database research, however, most of these genes had no direct relevance to eggshell calcification or calcium metabolism, expect for rs which located on the 3′utr of itpr gene. the detailed message of promising loci and the corresponding candidate genes are listed in table  table putative genes associated with eggshell quality gene symbola chr chromosome, aa amino acid, sift sorting intolerant from tolerant aidentification of the gene according to ensembl genes database 76 bsift is a program that predicts whether an amino acid substitution affects protein function. small values means deleterious amino acid change discussion it is known that eggshells are subject to quality loss along with the age process. in egg-type chicken production, there exists a trend to prolong the laying cycles, whereas the decline of eggshell quality for old hens proposes a substantial challenge for this development pattern. in the present study, we elucidated genomic architecture underlying the age-dependent dynamic eggshell quality, which has biological and practical significance. the qtl number detected for eggshell quality was limited in previous studies. the low to medium heritability of the eggshell trait revealed by our results and other study suggested the necessary use of large population for causal mutant identification. our f population consisting of hens is the largest population used for eggshell quality gwa analysis so far, and therefore the novel genomic region and loci revealed by the current study should be accurate and reliable. our gwa analysis for dynamic eggshell quality traits identified thousands of significant associations, that were much more than those screened by previous gwa studies in chicken. these significant mutations were almost in a same region with an extremely strong linkage disequilibrium status, which may result from the insufficient recombination events in the f segregation population. lots of qtl regions affecting esw, est and ess were discovered by previous linkage studies and mainly distributed on gga 1– and gga the promising genomic region from to  mb identified in the current study had no overlap with the previously reported regions in gga mainly on –  mb, –  mb, –  mb and –  mb. the significant snps in this region were distributed on independent genes. we tried to perform gene ontology and pathway analysis on these genes, whereas no significant go terms or pathways were enriched. this suggested there were not a series of functionally similar genes in this region to influence eggshell quality together. most snps might passively display significant associations for their linkages to a sort of real causal mutants on one or a few genes. hence, the phenotypic variance explained by all loci in this region was only slightly higher than single locus. it was notable that more associations were discovered for old hens than young hens, suggesting certain genetic variants were age-dependent. this was partially consistent with the other gwa studies, indicating no significant associations with both early and late shell quality. however, our results also proved the existence of common genetic variants consistently influencing eggshell quality from to weeks. six significant missense mutations in this genomic region were considered as the most important putative variants. after multivariate gwa analysis, only two loci, rs and rs on pik3c2g and itpr in gga remained significantly associated with dynamic eggshell quality traits. the loci of rs on pik3c2g could explain the most phenotypic variance of eggshell quality traits among the five missense mutations. hens with homozygotes of advantageously effective alleles could produce eggs with excellent ess, which decreased relatively within a narrow range along with the aging process. pik3c2g encoded proteins belonging to the phosphoinositide 3-kinase family, containing a lipid kinase catalytic domain as well as a c-terminal c domain, which acted as calcium-dependent phospholipid binding motifs. previous proteomic screens revealed that a high proportion of lipid-binding proteins abundantly existed in eggshell matrix such as extracellular fatty acid-binding protein, prosaposin and apolipoprotein d. considering the low abundance of lipids in eggshell, the existence of high abundance of lipid-binding proteins in eggshell matrix was perplexed. furthermore, one association analysis revealed another lipid-related gene, low-density lipoprotein receptor-related protein gene, as a new member of eggshell matrix protein, was significantly associated with eggshell traits. now in the current study pik3c2g possessing the c domain acting as lipid binding motif was also implicated in eggshell property. the role of lipid binding proteins in eggshell or during eggshell formation should be re-recognized. pik3c2g possessing the c domain could mediate translocation of proteins to lipid membranes, and also regulate protein-protein interactions in human and mammals. the interoperable matrix proteins and calcite together formed the bioceramic eggshell. it could be hypothesized that lipid binding proteins might act as carrier or modifier of matrix precursors, which mediate eggshell calcification process or act as frame proteins deposited into eggshell structure. consequently, pik3c2g was considered as candidate gene for eggshell quality. the gene of inositol,5-trisphosphate receptor type harboring the loci of rs was the positional and functional candidate gene for eggshell quality revealed by the current gwa analysis. the mutation of rs was a nonconservative substitution of serine by glycine, with the glycine-encoding allele being associated with stronger eggshell. a gene could be inactivated by a mutation either in a control site or in a coding region. so rs on 3′urt of itpr was also the susceptibility locus. itpr was known for its mediation in endoplasmic reticulum calcium release and inositol,5-trisphosphate could mobilize ca2+ from intracellular calcium stores to many types of cells. the existence of itpr in uterine epithelial cell of chicken had been verified, furthermore, the expression of itpr in uterus during eggshell calcification was significantly higher than that in magnum and duodenum which also possess active calcium metabolism. this provided the evidences that itpr play roles in the regulation of intra-cellular ca2+ transportation in uterus and contributed to the process of eggshell calcification. recently, a genome-wide association study in humans identified itpr as a susceptibility gene for kashin-beck disease which is a chronic osteochondropathy, mainly characterized by cartilage degeneration, cartilage matrix degradation, chondrocyte necrosis and apoptosis. this uncovered association with bone disease indicated that it might play a role in general mineralization processes. birds process medullary bone, a nonstructural type of woven bone, to act as a reservoir for the minerals required for shell calcification. our previous comparative proteomic analysis for uterine fluid and eggshell matrix proteins also identified that many bone-development related proteins were up-presented in strong eggshell group. up to date, there only existed one paper referred to the association of ion transporter genes with eggshell quality. they found one snp on a sodium channel gene had effects on eggshell strength, however, the phenotypic variance of ess it could explained was relatively low compared to that of itpr in the current study. all the above evidences suggested itpr should be a crucial and promising candidate gene relating to eggshell calcification as well as eggshell mechanical property. another missense mutation located on non-smc condensin i complex subunit g gene in gga was discovered in association with eggshell weight for young hens in our gwa analysis. many other studies reported multiple genomic regions containing this gene were identified to be associated with body weight and egg weight in chickens and many other growth traits in livestocks. the current study suggested that ncapg also involved in influencing eggshell weight. larger eggs generally owned heavier eggshell. hence we could not exclude the potential cause that the significant association of ncapg with esw was just due to its relevance to egg weight. nevertheless, we still considered ncapg as a candidate gene for its consistent association with eggshell weight from to week. previous studies hypothesized that eggshell organic matrix as a complicated mixture of proteins might play a regulatory role in assembly of the calcite zone with calcium carbonate to ultimately determine its strength, and many evidences had been proposed to support this, such as the different matrix protein concentrations in strong and weak eggshells. furthermore, many works conducted to investigate the associations between polymorphisms in genes encoding eggshell matrix proteins and eggshell quality traits. takahashi et al. conducted association analysis between ovocalyxin- gene haplotypes and eggshell quality traits in an f intercross and dunn et al. carried out association analysis between polymorphisms in eggshell organic matrix genes and eggshell quality measurements in two lines. these two studies found some significant associations, however, the loci could only explain limited phenotypic variances. our current study discovered no association between snps on eggshell matrix genes and eggshell quality, suggesting that the influences of eggshell matrix genes on eggshell quality were not caused by their nucleotide polymorphisms.  the dynamic eggshell quality traits at time points from onset of laying to weeks were collected and used for genome-wide association analysis with a 600 k high-density snp array. according to univariate and multivariate gwa analysis, we discovered a genomic region spanning from to  m in gga significantly associated with eggshell quality. ld and conditional analysis suggested this region were in extremely strong linkage disequilibrium status, especially for the ld block from to  mb that almost include half of the total significant loci. ultimately, three genes, pik3c2g, itpr and ncapg identified from three missense mutations were considered as promising candidate genes that implicated in dynamic eggshell quality. the homozygotes of advantageously effective alleles on pik3c2g and itpr possessed the best eggshell quality and could partly counteract the negative effect of aging process. these promising loci and genes could be helpful to engineer practical breeding programs for the improvement of eggshell quality for old hens to meet the need of prolonging the laying cycle. the new findings also have greatly advanced our understanding of the genetic basis underlying eggshell quality. 
however, the genetic architecture underlying the dynamic eggshell quality remains elusive. identification of the promising region as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic eggshell quality and has the practical significance in breeding program for the improvement of eggshell quality, especially at the later part of laying cycle. most significant loci were in a region spanning from to  mb of chromosome, which together account for  ~   % of the phenotypic variance for esw from to weeks of age,  ~   % and  ~   % for est and ess from to weeks of age. the homozygotes of advantageously effective alleles on pik3c2g and itpr possessed the best eggshell quality and could partly counteract the negative effect of aging process. ultimately, two missense snps in gga and one in gga were considered as promising loci on three independent genes including itpr pik3c2g, and ncapg. according to linkage disequilibrium and conditional analysis, the significant snps in this region were in extremely strong linkage disequilibrium status. ncapg had certain effect on eggshell quality for young hens. here, we measured eggshell quality traits, including eggshell weight, eggshell thickness and eggshell strength at time points from onset of laying to weeks of age and conducted comprehensive genome-wide association studies in 1534 f hens derived from reciprocal crosses between white leghorn and dongxiang chickens. gwaseggshell qualityage-dependent traitssnp-based heritabilitychickenissue-copyright-statement© the author 2015 eleven independent univariate genome-wide screens for each trait totally identified and significant associations with esw, est and ess, respectively. esws at all ages exhibited moderate snp-based heritability estimates, while the estimates for est and ess were relatively low. eggshell is subject to quality loss with aging process of laying hens, and damaged eggshells result in economic losses of eggs.
 eggshell is subject to quality loss with aging process of laying hens, and damaged eggshells result in economic losses of eggs. however, the genetic architecture underlying the dynamic eggshell quality remains elusive. here, we measured eggshell quality traits, including eggshell weight, eggshell thickness and eggshell strength at time points from onset of laying to weeks of age and conducted comprehensive genome-wide association studies in 1534 f hens derived from reciprocal crosses between white leghorn and dongxiang chickens.  esws at all ages exhibited moderate snp-based heritability estimates, while the estimates for est and ess were relatively low. eleven independent univariate genome-wide screens for each trait totally identified and significant associations with esw, est and ess, respectively. most significant loci were in a region spanning from to  mb of chromosome, which together account for  ~   % of the phenotypic variance for esw from to weeks of age,  ~   % and  ~   % for est and ess from to weeks of age. according to linkage disequilibrium and conditional analysis, the significant snps in this region were in extremely strong linkage disequilibrium status. ultimately, two missense snps in gga and one in gga were considered as promising loci on three independent genes including itpr pik3c2g, and ncapg. the homozygotes of advantageously effective alleles on pik3c2g and itpr possessed the best eggshell quality and could partly counteract the negative effect of aging process. ncapg had certain effect on eggshell quality for young hens.  identification of the promising region as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic eggshell quality and has the practical significance in breeding program for the improvement of eggshell quality, especially at the later part of laying cycle. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gwaseggshell qualityage-dependent traitssnp-based heritabilitychickenissue-copyright-statement© the author 2015 