BACKGROUND
residual feed intake  is an effective indicator for the feed utilization efficiency of animals.
low rfi selection enables a lower energy intake without sacrificing growth performance because it is independent of the metabolic body weight and daily gain regardless of the phenotype or genetic level  <cit> .
the feed conversion rate  is another synthetic trait that can optimize energy usage and is negatively correlated with growth traits  <cit> .
chinese consumers prefer quality chickens  with a long feeding time and excellent meat quality but focus less on body weight at the market.
therefore, rfi selection is more applicable to non-fast growing chickens for the genetic improvement of energy metabolism.
to date, up to  <dig> quantitative trait loci  have been reported to have significant associations with chicken feeding traits including dry matter digestibility, dry matter intake, feeding efficiency, feed intake, rfi and fcr, of which  <dig> were associated with rfi .
however, the above rfi-related qtls are difficult to be applied to quality chickens due to the differences on breeds and trait measurement time.
genome-wide association studies , at first, are used to screen candidate markers associated with human diseases  <cit> .
with the continuous improvement and reduced cost of genotyping technology, this technique is being more widely applied to animal breeding and genetics.
gwas-related applications in rfi are primarily concentrated on beef cattle  and pigs  and have been applied to laying hens  <cit>  and broilers  <cit> .
the development of the high density 600 k genotyping array may provide technical assistance for the identification of causative and credible single nucleotide polymorphisms  that affect the chicken rfi  <cit> .
the transcriptome is a full set of the rnas transcribed in the cell at a certain developmental phase or under a specific stress condition and possesses spatial and temporal expression characteristics  <cit> .
rna sequencing can be a gateway to success in identifying differentially expressed genes , discovering alternative splicing events, and conducting studies on gene evolution .
previous studies showed that the rfi was so complex that all relevant genes might not operate in a single physiological pathway  <cit> .
the present study combined gwas with transcriptome sequencing to identify distinct genes or regions that affected the chicken rfi.
our results lay the foundation for improvements in energy efficiency and reductions in feed cost.
RESULTS
growth characteristics and genetic parameters
the pure line n <dig> belongs to the dwarfism yellow-plumage chicken with a medium growth speed.
at the beginning of the feeding trial , the average body weights of the male and female chickens were  <dig>  g and  <dig>  g, respectively.
when the trial was finished , the male chickens reached  <dig>  g and the female chickens reached  <dig>  g. the average daily feed intakes  of the male and female chickens recorded by the electronic feeding station were  <dig>  g and  <dig>  g, respectively.
the linear regression equation well described the changes in body weight at different ages.
the average r <dig> across all birds was  <dig>  %.
only four birds had an r <dig> lower than 80 % , suggesting that the estimates for average daily gain  and mid-test metabolic body weight  for each bird were reliable.fig.
<dig> distributional r <dig> for regression of body weight against the day of the test for each chicken.
the vertical ordinate denotes the r <dig> for the growth rate
the test of fixed effects showed that the gender effect on rfi was not significant , but the pen effect was significant .
one explanation for this result may be that the chickens were already separated based on gender when they were arranged in different pens, thereby turning the nested gender effect into the pen effect.
next, we compared the growth traits between the birds with the highest- and lowest-ranked rfis .
no significant difference was found for any growth trait, which was similar to the results obtained in cattle  <cit> .
however, the adfi for the lowest rfi-ranked birds was 39 g lower than the adfi for the highest rfi-ranked birds .table  <dig> growth characteristics  of 10 % of the chickens with the lowest and highest residual feed intake rankings
traitsa
p-valueb
a
rfi residual feed intake, adfi average daily feed intake, adg average daily gain, mbw mid-test body weight, fcr feed conversion rate
b
p-value obtained by t-test
the heritability estimate for the rfi from  <dig> to  <dig> d of age was  <dig>  , which fell into the  <dig>  to  <dig>  range reported for other populations  <cit> .
this result implied that the rfi was under a moderate level of genetic control.
phenotypic rfi selection provides a method to reduce feed intake without affecting either the adg  or mmbw .
however, rfi selection had a few consequences over the adg if only the genetic contribution was considered , which was consistent with previous reports  <cit> .
the positive genetic correlation between rfi and fcr found in our population  was higher than the correlation reported in other domestic animals  <cit> .table  <dig> genetic parameter estimation for the growth and feeding traits
traitsc
apearson correlation
bgenetic correlation
c
rfi residual feed intake, adfi average daily feed intake, adg average daily gain, mbw mid-test body weight, fcr feed conversion rate
d number of samples for estimating heritability
genome-wide association study
the original rfi values calculated from  <dig> individuals were examined for compliance with normality prior to the gwas using the anderson-darling test.
the results showed that the initial distribution deviated from the normal distribution  .
after johnson transformation, the phenotypic data were subjected to statistical analysis .fig.
<dig> probability graph of residual feed intake
because the kinship of all tested individuals could not be entirely eliminated when the ibs matrix was introduced into the mixed model, the genomic control inflation/deflation parameter  was introduced  <cit> .
in total,  <dig> snps were significantly associated with the rfi with p-values below the threshold  after correction by a λ equal to  <dig>  ; these snps accounted for  <dig>  % of the additive genetic variance.
these significant points corresponded to  <dig> known genes and one unannotated gene in the chicken genome: slc17a <dig>  cobl, pcdh <dig>  jakmip <dig>  zfyve <dig>  ppp1r <dig>  sept <dig>  rybp, pdzrn <dig>  chl <dig>  uts2r, zmpste <dig>  syt <dig> and loc <dig> .fig.
<dig> manhattan plot of the snp additive effects on residual feed intake  in chickens.
the green line was the genome-wide significance threshold
p-valuec
aphysical position
badditive effect of allele b
c
p-value corrected for inflation factor λ, *fdr ≤10 %, ** fdr ≤5 %
dminor allele frequency
ethe proportion of the phenotypic variance accounted by the snp
these snps were widely distributed on eight chicken  chromosomes .
gga <dig>  gga <dig> and gga <dig> possessed only one significant snp each.
two of the significant snps were located in the 3’ untranslated region  and downstream of slc17a <dig> on gga <dig>  two were located within a  <dig> – <dig>  mb region on gga <dig>  three were located within a  <dig> – <dig>  mb region on gga <dig>  five were located on gga <dig>  notably, more than half of the total significant snps were located within a 1 mb narrow region  on gga <dig>
this narrow region contained  <dig> tested snps around eight genes.
we observed  <dig> haplotype blocks extending from  <dig> to 64 kb .
the significant snps were entirely located within the blocks with the exception of snp rs <dig>  three significant points in the rybp introns  had identical additive effects because they were in a state of complete linkage.
six haplotypes were found in a block determined by the snps rs <dig> and rs312899555; “gatga” occurred more commonly in the other three non-significant snps with a frequency of  <dig>  %.
four haplotypes were found in another block determined by the snp rs313947867; “gggc” was the most common haplotype in the other three non-significant snps with a frequency of 35 %.
five significant snps in complete linkage were found in the introns of pdzrn <dig>  although they were positioned in two different blocks.fig.
<dig> ensembl genes on the region from  <dig>  to  <dig>  mb on chicken chromosome  <dig> and haplotype blocks constructed using significant rfi-related snps
the proportion of phenotypic variance explained by rs <dig> in the intron of jakmip <dig> was highest among the significant snps.
because the original rfi variable was transformed into the normal distribution data, the increment of the additive effect accompanied by the minor allele could not accurately reflect its impact on the change in rfi.
in contrast, the direction of the additive effect could be used to judge whether the minor allele would be beneficial to the rfi.
the results showed that nine wild-type mutations were in favor of decreasing the chicken’s rfi value.
the chip used in the present study had a very high density.
the average interval between two snps was  <dig>  kb, which resulted in little chance for recombination among neighboring snps gathered in a limited range of the chromosome.
linkage disequilibrium  analysis was performed to determine the extent to which the causative snp could generate linkage to the significant snp.
the pairwise ld measured by r <dig> values for the present population was calculated for gga <dig>  where the most significant snps were concentrated.
the results showed that the ld level was high  at short distances, slightly decreased  when the distances increased to 40–60 kb and low  when the distance increased to 80–100 kb.
similar trends were found in gga <dig> .
therefore, 50 kb was accepted as a reasonable distance to cause moderate ld between two snps, and we captured all genes distributed on the 50 kb flanking regions of the significant snps for go analysis.fig.
<dig> relationship between the physical distance and ld values between snp markers in the quality chickens used in the present study
the results from the first level classification revealed that  <dig> go terms in molecular function,  <dig> go terms in biological process and  <dig> of go terms in cellular component corresponded to the input genes .
a deeper analysis was conducted in the molecular function go terms to obtain more information.
protein binding  was reflected by eight different genes and might be the major molecular function that differed in rfi-divergent birds, followed by zinc ion binding , calcium ion binding , and atp binding .
protein binding was significant, including the synthesis of a peptides from amino acid molecules and the formation of a high-grade protein structure by combining polypeptide chains.
this result indicated that the difference in protein binding was associated with the rapid growth of the experimental birds, specifically muscle development.
the chief role of the calcium ion was to compromise potential on two sides of the cell membranes and to maintain muscle contraction and relaxation, indicating that differences in motor skills would occur in rfi-divergent birds.
therefore, differences in atp binding should not be overlooked.
atp is the primary source of fuel for every biochemical and physiological process and will induce metabolic disorders in the event of an exception.
rna sequencing of the transcriptome
read mapping
rna sequencing was performed in four individuals, as follows: two chickens with the highest rfi values  and two with the lowest rfi values .
among the four samples, h <dig> had the lowest mapping rate  of approximately  <dig>  % compared to the rates above 90 % for the other three samples .
some of these reads had more than one matching sequence in the genome; these reads were removed from the subsequent analysis.
the clean reads were evenly distributed across all chromosomes.
based on the annotation analysis of the clean reads mapped to the genome, we found that 61 % of these reads were derived from exons,  <dig> – <dig>  % were from intergenic regions, and only a small number were derived from splicing and intronic regions.table  <dig> statistics for read mapping
total readsa
total mappedb
multiple mappedc
uniquely mappedd
reads map to “+”e
reads map to “−”f
atotal reads: the number of clean reads
btotal mapped: the number of clean reads that could be mapped to the chicken genome
cmultiple mapped: the number of clean reads corresponding to a plurality of locations in the genome
duniquely mapped: the number of clean reads corresponding to a unique location in the genome
ereads map to “+”: reads mapped to the sense strand
freads map to “−”: reads mapped to the antisense strand
deg analysis
four samples were classed into two groups based on their rfi rankings prior to the deg analysis.
the average expression of identical genes in the high-rfi group was compared with the low-rfi group to screen for degs.
biological replicates of the expressed genes had a high correlation coefficient  between the high-rfi group and low-rfi group .
as a generalized representation index, rpkm  was used to denote each transcript’s expression quantity  <cit> .
after bioinformatics assessment and comparison,  <dig> differentially expressed transcripts were identified between the high- and low-rfi groups that corresponded to  <dig> known genes.
a total of  <dig> of the degs were up-regulated in the low-rfi group and the rest were down-regulated compared with high-rfi group .
interestingly, three genes  were only expressed in the low-rfi group.
the expression of mir- <dig> was significantly lower in the low-rfi group than in the high-rfi group with a fold change of eight.
in contrast, the expression of pck <dig> was significantly higher in the low-rfi group than in the high-rfi group with a fold change of  <dig>
mirdb  and targetscan  are two effective websites for microrna  target prediction.
based on their prediction results, mir-122-5p and mir-122-3p were predicted to act on  <dig> target genes.
three targets  were found to be degs.
however, these genes were up-regulated in the low-rfi group, which was not consistent with the negative regulatory interaction between the mirna and its target.
mir-6705-5p was predicted to act on  <dig> target genes.
nine targets  were found to be degs.
except for lpl, the other eight genes were up-regulated in the low-rfi group, indicating they might be negatively regulated by mir-6705-5p.
of note, the expression of col3a <dig> and cav <dig> could be impacted by both the mir-122- <dig> and mir- <dig>
hepatic pck <dig> expression was drastically increased by 28-fold compared to its normal expression level.
similarly, the subcutaneous, intercellular, and abdominal fat contents were increased when a simple orchiectomy was implemented in capons, implying negative regulation of hepatic pck <dig> expression and abdominal fat content by testosterone  <cit> .
discussion
in koch’s model, b2bw <dig>  is normally used as metabolic energy  for maintenance.
however, b2bw <dig>  is most likely to represent only the me maintenance output for basal metabolism.
to determine the maintenance requirement, two non-determinative factors  should be considered simultaneously.
in the present study, rfi was phenotypically independent of mmbw  and adg , indicating that there was no difference in the use of the me for maintenance of the basal metabolism and for growth between high and low rfi individuals  <cit> .
in reality, high-rfi birds tried to replenish their energy by eating more; thus, the redundant energy might be exploited by stress or physical activity  <cit> .
we speculated that the low-rfi birds had less governable overall energy to expend on stress and physical activity.
in the present study, the low-rfi birds spent less time eating  , indicating that they reduced the me demand at the expense of less physical activity related to feed intake.
four significant snps on gga <dig> and gga <dig> in the present study did fall into the reported qtls for chicken rfi  <cit> .
however, only the significant snps on gga <dig> passed chromosome-wise fdr threshold of 5 %, probably because of limited sample size resulting in low power to detect a qtl by testing the marker effect.
another possible cause was the low rfi phenotypic variability in sex-linked dwarf chicken used in the present study.
the rfi values of  <dig> individuals ranged from − <dig>  g to  <dig>  g. the standard deviation of the rfi was simply  <dig>  g, which was much less than that observed in normal meat-type chickens  <cit> .
in the present study, we identified eight genes located in a 1 mb narrow region, and almost all of the significant points in gga <dig> were located in introns.
the snps in the introns could play negative, positive, or bidirectional regulatory roles in gene expression and affect alternative splicing  <cit> .
pdzrn <dig> was reported to exert a negative effect on lipogenesis.
in mouse 3 t3-l <dig> preadipocytes, up-regulation of stat5b and c/ebpβ was observed in response to pdzrn <dig> silencing, resulting in increased expression of pparγ at both the mrna and protein levels and the promotion of 3 t3-l <dig> cell differentiation into adipocytes  <cit> .
as a candidate gene for human schizophrenia and mental deficiency, chl <dig> is relevant to learning behavior and reorganization of the frame of thinking.
the chl1-deficient mice displayed reduced enthusiasm for fresh food hunting and a blockage in social contact that was attributed to chaos in the neural circuits that connected the brain’s limbic system to the cerebral cortex  <cit> .
jakmip <dig> was of special concern because its intronic snp rs <dig> explained the highest phenotypic variance.
this gene was reported to be associated with microtubule polymers and to participate in cytoskeleton rearrangement, cell polarization, and intracellular trafficking  <cit> .
these findings combined with the implications of the go analysis provided hints that rfi-related genes improved energy utilization efficiency by adjusting cellular procedural activities, thereby assisting muscle and neural development and growth and eventually improving and enhancing energy efficiency.
transcriptome sequencing helped identify the degs causing the diversity in the rfi phenotypes.
in the absence of mrna expression, we speculate that there should be a role for cckar in the constant energy use.
indeed, cckar was reported to be associated with appetite control  <cit> .
cck is a hormone that causes gallbladder contractions and enables the promotion of trypsin secretion  <cit> .
central cck could be motivated by a combination of endogenous cck and its receptor cckar to trigger a feeling of satiety.
indeed, an increased feed intake was detected in cckar-deficient mice  <cit> , and the risk variants of porcine cckar were significantly correlated with feeding traits  <cit> .
the effect of the cck gene might be weakened by a lack of its receptor in high-rfi individuals, resulting in increased feed intake.
the degs were consigned to six different gene-interaction networks predicted by ipa .
the networks were more comprehensive and detailed when combined with the function of homologous genes from humans and mice.
for this reason, they provided redundant information that could not be applied to chickens, such as cancer or cardiac failure; thus, the related gene functions were simply suggestive in poultry.
however, these networks provided important clues to improve the energy balance from the perspectives of genetics, nutrition and bio-pharmaceuticals.
low rfi selection made dramatic changes in some biological pathways possible .
for example, the cell death and apoptosis of tumor cell lines, activation of antigen presenting cells  and cell viability of kidney cell lines were decreased during low rfi selection.
additionally, chickens with low rfi were more prone to vascular lesions.
every change was a result of the mutual action of degs.table  <dig> predictable changes in biological functioning based on deg analysis in low-rfi individuals
apoptosis and apc viability warranted attention because they were tightly linked to immunity.
apoptosis was effectively under the control of genes.
when low-rfi birds suffer from avian leukosis virus, marek’s disease or reticuloendotheliosis, related degs might be responsible for blocking the energy supply to tumor cells, thereby slowing apoptosis and accelerating tumor formation.
apcs are capable of ingesting and processing pathogenic microorganisms by phagocytosis or pinocytosis, which in turn produces peptide fragments that contain antigenicity domains  <cit> .
decreased activation of apcs in low-rfi birds might cause a delay in the immune response or a reduced ability to kill pathogens.
hsp90b <dig> is an important immune protein that is involved in cell protection under heat stress.
increased hsp90b expression in response to heat stress causes various reductions in cell damage and even repairs damaged proteins  <cit> .
the reduced hsp90b <dig> expression in low-rfi birds signified a weaker ability to resist the effect of environmental factors compared to high-rfi birds.
additionally, the changes in some degs were found to function in the regulation of fat metabolism .
for low-rfi birds,  <dig> degs were predicted to facilitate the concentration of lipids,  <dig> were predicted to play a pivotal role in the activation of lipid synthesis, and  <dig> were associated with the activation of fatty acid metabolism.
although a few degs had inhibitory effects, the above three specific physiological functions seemed to be enhanced overall.
the results in the present study showed that low-rfi birds could achieve greater synthetic metabolism of lipids compared to high-rfi birds, whereas the results of another study showed that they displayed weaker catabolic abilities  <cit> .
the faster anabolism of fat than catabolism in low-rfi birds might cause abnormal storage of fat and a decrease in heat release.fig.
<dig> predictable changes in fat metabolism based on deg analysis in low-rfi individuals
among the lipometabolism-related degs, pck <dig> was particularly noteworthy because it was remarkably increased in the low-rfi group.
the pck <dig> gene is associated with obesity, insulin resistance, and type ii diabetes in mammals .
abdominal fat content was assumed to be positively correlated with pck <dig> mrna expression in birds  <cit> .
thus, high pck <dig> expression in low-rfi birds might signal a concurrent increase in the abdominal fat mass.
interestingly, ar expression was significantly lower in the low-rfi birds than in the high-rfi birds.
the interaction of androgen with its receptor  was closely related to bone metabolism in birds  <cit> .
the ar in the cytoplasm enters the nucleus by integrating with androgen and then binding to the androgen responsive element, leading to the activation of androgen  <cit> .
low ar expression interferes with the positive physiological effect of androgen, leading to high pck <dig> expression.
a systematic analysis of the gwas and rna sequencing results was performed.
the results showed there was no overlap of the genes within the 50 kb flanking region containing the significant snps detected by gwas and any deg detected by rna sequencing.
one explanation is that the actual linkage distance between the qtls and the significant snp is larger than 50 kb.
however, we stopped measuring the rfi when the chickens were  <dig> d of age, whereas the liver sample collection was performed at  <dig> d of age.
during this time, the rfi ranking may have slightly changed, resulting in a mismatch.
nonetheless, when the search scope was broadened  <cit>  we still found that some potential genes within the 500-kb flanking region of the significant rfi-related snps in gwas were identified as degs in chickens with divergent rfis, including mgp, hist1h <dig>  hist1h2a4l <dig> and oc <dig> near two snps  in gga <dig>  nr0b <dig> near the snp rs <dig>  per <dig> near the snp rs <dig>  st6galnac <dig> near the snp rs <dig>  and g0s <dig> near the snp rs <dig>  notably, the concurrent genes involved in gwas and rna sequencing played critical roles in a wide spectrum of biological processes.
therefore, their relationships with rfi need to be verified.
CONCLUSIONS
the heritability of rfi during the period from  <dig> to  <dig> d of age was moderate.
the rfi was particularly affected by  <dig> significant snps that could explain  <dig>  % of the additive genetic variance.
seventeen rfi-related snps were located in a 1 mb region  of gga <dig>  this region was identified as a key candidate region affecting the energy utilization efficiency of chickens because it contained genes associated with lipogenesis, social behavior, and immunity.
although they obtained less metabolic energy, low-rfi birds maintained normal growth that was comparable to that of the high-rfi birds by allocating less energy to cell activity, the immune response, and physical activity in relation to eating and thus optimizing the use of the limited resources.
nevertheless, low-rfi selection cannot mobilize spare energy to withstand environmental stress and causes potential health risks that should not be ignored.
at the same time, the low-rfi birds tended to become fatter.

 residual feed intake is an effective indicator for the feed utilization efficiency of animals. low rfi selection enables a lower energy intake without sacrificing growth performance because it is independent of the metabolic body weight and daily gain regardless of the phenotype or genetic level. the feed conversion rate is another synthetic trait that can optimize energy usage and is negatively correlated with growth traits. chinese consumers prefer quality chickens with a long feeding time and excellent meat quality but focus less on body weight at the market. therefore, rfi selection is more applicable to non-fast growing chickens for the genetic improvement of energy metabolism. to date, up to quantitative trait loci have been reported to have significant associations with chicken feeding traits including dry matter digestibility, dry matter intake, feeding efficiency, feed intake, rfi and fcr, of which were associated with rfi. however, the above rfi-related qtls are difficult to be applied to quality chickens due to the differences on breeds and trait measurement time. genome-wide association studies, at first, are used to screen candidate markers associated with human diseases. with the continuous improvement and reduced cost of genotyping technology, this technique is being more widely applied to animal breeding and genetics. gwas-related applications in rfi are primarily concentrated on beef cattle and pigs and have been applied to laying hens and broilers. the development of the high density 600 k genotyping array may provide technical assistance for the identification of causative and credible single nucleotide polymorphisms that affect the chicken rfi. the transcriptome is a full set of the rnas transcribed in the cell at a certain developmental phase or under a specific stress condition and possesses spatial and temporal expression characteristics. rna sequencing can be a gateway to success in identifying differentially expressed genes, discovering alternative splicing events, and conducting studies on gene evolution. previous studies showed that the rfi was so complex that all relevant genes might not operate in a single physiological pathway. the present study combined gwas with transcriptome sequencing to identify distinct genes or regions that affected the chicken rfi. our results lay the foundation for improvements in energy efficiency and reductions in feed cost.  growth characteristics and genetic parameters the pure line n belongs to the dwarfism yellow-plumage chicken with a medium growth speed. at the beginning of the feeding trial, the average body weights of the male and female chickens were g and g, respectively. when the trial was finished, the male chickens reached g and the female chickens reached g. the average daily feed intakes of the male and female chickens recorded by the electronic feeding station were g and g, respectively. the linear regression equation well described the changes in body weight at different ages. the average r across all birds was  %. only four birds had an r lower than 80 %, suggesting that the estimates for average daily gain and mid-test metabolic body weight for each bird were reliable.fig. distributional r for regression of body weight against the day of the test for each chicken. the vertical ordinate denotes the r for the growth rate the test of fixed effects showed that the gender effect on rfi was not significant, but the pen effect was significant. one explanation for this result may be that the chickens were already separated based on gender when they were arranged in different pens, thereby turning the nested gender effect into the pen effect. next, we compared the growth traits between the birds with the highest- and lowest-ranked rfis. no significant difference was found for any growth trait, which was similar to the results obtained in cattle. however, the adfi for the lowest rfi-ranked birds was 39 g lower than the adfi for the highest rfi-ranked birds.table growth characteristics of 10 % of the chickens with the lowest and highest residual feed intake rankings traitsa p-valueb a rfi residual feed intake, adfi average daily feed intake, adg average daily gain, mbw mid-test body weight, fcr feed conversion rate b p-value obtained by t-test the heritability estimate for the rfi from to d of age was, which fell into the to range reported for other populations. this result implied that the rfi was under a moderate level of genetic control. phenotypic rfi selection provides a method to reduce feed intake without affecting either the adg or mmbw. however, rfi selection had a few consequences over the adg if only the genetic contribution was considered, which was consistent with previous reports. the positive genetic correlation between rfi and fcr found in our population was higher than the correlation reported in other domestic animals.table genetic parameter estimation for the growth and feeding traits traitsc apearson correlation bgenetic correlation c rfi residual feed intake, adfi average daily feed intake, adg average daily gain, mbw mid-test body weight, fcr feed conversion rate d number of samples for estimating heritability genome-wide association study the original rfi values calculated from individuals were examined for compliance with normality prior to the gwas using the anderson-darling test. the results showed that the initial distribution deviated from the normal distribution. after johnson transformation, the phenotypic data were subjected to statistical analysis.fig. probability graph of residual feed intake because the kinship of all tested individuals could not be entirely eliminated when the ibs matrix was introduced into the mixed model, the genomic control inflation/deflation parameter was introduced. in total, snps were significantly associated with the rfi with p-values below the threshold after correction by a λ equal to; these snps accounted for  % of the additive genetic variance. these significant points corresponded to known genes and one unannotated gene in the chicken genome: slc17a cobl, pcdh jakmip zfyve ppp1r sept rybp, pdzrn chl uts2r, zmpste syt and loc.fig. manhattan plot of the snp additive effects on residual feed intake in chickens. the green line was the genome-wide significance threshold p-valuec aphysical position badditive effect of allele b c p-value corrected for inflation factor λ, *fdr ≤10 %, ** fdr ≤5 % dminor allele frequency ethe proportion of the phenotypic variance accounted by the snp these snps were widely distributed on eight chicken chromosomes. gga gga and gga possessed only one significant snp each. two of the significant snps were located in the 3’ untranslated region and downstream of slc17a on gga two were located within a –  mb region on gga three were located within a –  mb region on gga five were located on gga notably, more than half of the total significant snps were located within a 1 mb narrow region on gga this narrow region contained tested snps around eight genes. we observed haplotype blocks extending from to 64 kb. the significant snps were entirely located within the blocks with the exception of snp rs three significant points in the rybp introns had identical additive effects because they were in a state of complete linkage. six haplotypes were found in a block determined by the snps rs and rs312899555; “gatga” occurred more commonly in the other three non-significant snps with a frequency of  %. four haplotypes were found in another block determined by the snp rs313947867; “gggc” was the most common haplotype in the other three non-significant snps with a frequency of 35 %. five significant snps in complete linkage were found in the introns of pdzrn although they were positioned in two different blocks.fig. ensembl genes on the region from to  mb on chicken chromosome and haplotype blocks constructed using significant rfi-related snps the proportion of phenotypic variance explained by rs in the intron of jakmip was highest among the significant snps. because the original rfi variable was transformed into the normal distribution data, the increment of the additive effect accompanied by the minor allele could not accurately reflect its impact on the change in rfi. in contrast, the direction of the additive effect could be used to judge whether the minor allele would be beneficial to the rfi. the results showed that nine wild-type mutations were in favor of decreasing the chicken’s rfi value. the chip used in the present study had a very high density. the average interval between two snps was kb, which resulted in little chance for recombination among neighboring snps gathered in a limited range of the chromosome. linkage disequilibrium analysis was performed to determine the extent to which the causative snp could generate linkage to the significant snp. the pairwise ld measured by r values for the present population was calculated for gga where the most significant snps were concentrated. the results showed that the ld level was high at short distances, slightly decreased when the distances increased to 40–60 kb and low when the distance increased to 80–100 kb. similar trends were found in gga. therefore, 50 kb was accepted as a reasonable distance to cause moderate ld between two snps, and we captured all genes distributed on the 50 kb flanking regions of the significant snps for go analysis.fig. relationship between the physical distance and ld values between snp markers in the quality chickens used in the present study the results from the first level classification revealed that go terms in molecular function, go terms in biological process and of go terms in cellular component corresponded to the input genes. a deeper analysis was conducted in the molecular function go terms to obtain more information. protein binding was reflected by eight different genes and might be the major molecular function that differed in rfi-divergent birds, followed by zinc ion binding, calcium ion binding, and atp binding. protein binding was significant, including the synthesis of a peptides from amino acid molecules and the formation of a high-grade protein structure by combining polypeptide chains. this result indicated that the difference in protein binding was associated with the rapid growth of the experimental birds, specifically muscle development. the chief role of the calcium ion was to compromise potential on two sides of the cell membranes and to maintain muscle contraction and relaxation, indicating that differences in motor skills would occur in rfi-divergent birds. therefore, differences in atp binding should not be overlooked. atp is the primary source of fuel for every biochemical and physiological process and will induce metabolic disorders in the event of an exception. rna sequencing of the transcriptome read mapping rna sequencing was performed in four individuals, as follows: two chickens with the highest rfi values and two with the lowest rfi values. among the four samples, h had the lowest mapping rate of approximately  % compared to the rates above 90 % for the other three samples. some of these reads had more than one matching sequence in the genome; these reads were removed from the subsequent analysis. the clean reads were evenly distributed across all chromosomes. based on the annotation analysis of the clean reads mapped to the genome, we found that 61 % of these reads were derived from exons, –  % were from intergenic regions, and only a small number were derived from splicing and intronic regions.table statistics for read mapping total readsa total mappedb multiple mappedc uniquely mappedd reads map to “+”e reads map to “−”f atotal reads: the number of clean reads btotal mapped: the number of clean reads that could be mapped to the chicken genome cmultiple mapped: the number of clean reads corresponding to a plurality of locations in the genome duniquely mapped: the number of clean reads corresponding to a unique location in the genome ereads map to “+”: reads mapped to the sense strand freads map to “−”: reads mapped to the antisense strand deg analysis four samples were classed into two groups based on their rfi rankings prior to the deg analysis. the average expression of identical genes in the high-rfi group was compared with the low-rfi group to screen for degs. biological replicates of the expressed genes had a high correlation coefficient between the high-rfi group and low-rfi group. as a generalized representation index, rpkm was used to denote each transcript’s expression quantity. after bioinformatics assessment and comparison, differentially expressed transcripts were identified between the high- and low-rfi groups that corresponded to known genes. a total of of the degs were up-regulated in the low-rfi group and the rest were down-regulated compared with high-rfi group. interestingly, three genes were only expressed in the low-rfi group. the expression of mir- was significantly lower in the low-rfi group than in the high-rfi group with a fold change of eight. in contrast, the expression of pck was significantly higher in the low-rfi group than in the high-rfi group with a fold change of mirdb and targetscan are two effective websites for microrna target prediction. based on their prediction results, mir-122-5p and mir-122-3p were predicted to act on target genes. three targets were found to be degs. however, these genes were up-regulated in the low-rfi group, which was not consistent with the negative regulatory interaction between the mirna and its target. mir-6705-5p was predicted to act on target genes. nine targets were found to be degs. except for lpl, the other eight genes were up-regulated in the low-rfi group, indicating they might be negatively regulated by mir-6705-5p. of note, the expression of col3a and cav could be impacted by both the mir-122- and mir- hepatic pck expression was drastically increased by 28-fold compared to its normal expression level. similarly, the subcutaneous, intercellular, and abdominal fat contents were increased when a simple orchiectomy was implemented in capons, implying negative regulation of hepatic pck expression and abdominal fat content by testosterone. discussion in koch’s model, b2bw is normally used as metabolic energy for maintenance. however, b2bw is most likely to represent only the me maintenance output for basal metabolism. to determine the maintenance requirement, two non-determinative factors should be considered simultaneously. in the present study, rfi was phenotypically independent of mmbw and adg, indicating that there was no difference in the use of the me for maintenance of the basal metabolism and for growth between high and low rfi individuals. in reality, high-rfi birds tried to replenish their energy by eating more; thus, the redundant energy might be exploited by stress or physical activity. we speculated that the low-rfi birds had less governable overall energy to expend on stress and physical activity. in the present study, the low-rfi birds spent less time eating, indicating that they reduced the me demand at the expense of less physical activity related to feed intake. four significant snps on gga and gga in the present study did fall into the reported qtls for chicken rfi. however, only the significant snps on gga passed chromosome-wise fdr threshold of 5 %, probably because of limited sample size resulting in low power to detect a qtl by testing the marker effect. another possible cause was the low rfi phenotypic variability in sex-linked dwarf chicken used in the present study. the rfi values of individuals ranged from −  g to  g. the standard deviation of the rfi was simply  g, which was much less than that observed in normal meat-type chickens. in the present study, we identified eight genes located in a 1 mb narrow region, and almost all of the significant points in gga were located in introns. the snps in the introns could play negative, positive, or bidirectional regulatory roles in gene expression and affect alternative splicing. pdzrn was reported to exert a negative effect on lipogenesis. in mouse 3 t3-l preadipocytes, up-regulation of stat5b and c/ebpβ was observed in response to pdzrn silencing, resulting in increased expression of pparγ at both the mrna and protein levels and the promotion of 3 t3-l cell differentiation into adipocytes. as a candidate gene for human schizophrenia and mental deficiency, chl is relevant to learning behavior and reorganization of the frame of thinking. the chl1-deficient mice displayed reduced enthusiasm for fresh food hunting and a blockage in social contact that was attributed to chaos in the neural circuits that connected the brain’s limbic system to the cerebral cortex. jakmip was of special concern because its intronic snp rs explained the highest phenotypic variance. this gene was reported to be associated with microtubule polymers and to participate in cytoskeleton rearrangement, cell polarization, and intracellular trafficking. these findings combined with the implications of the go analysis provided hints that rfi-related genes improved energy utilization efficiency by adjusting cellular procedural activities, thereby assisting muscle and neural development and growth and eventually improving and enhancing energy efficiency. transcriptome sequencing helped identify the degs causing the diversity in the rfi phenotypes. in the absence of mrna expression, we speculate that there should be a role for cckar in the constant energy use. indeed, cckar was reported to be associated with appetite control. cck is a hormone that causes gallbladder contractions and enables the promotion of trypsin secretion. central cck could be motivated by a combination of endogenous cck and its receptor cckar to trigger a feeling of satiety. indeed, an increased feed intake was detected in cckar-deficient mice, and the risk variants of porcine cckar were significantly correlated with feeding traits. the effect of the cck gene might be weakened by a lack of its receptor in high-rfi individuals, resulting in increased feed intake. the degs were consigned to six different gene-interaction networks predicted by ipa. the networks were more comprehensive and detailed when combined with the function of homologous genes from humans and mice. for this reason, they provided redundant information that could not be applied to chickens, such as cancer or cardiac failure; thus, the related gene functions were simply suggestive in poultry. however, these networks provided important clues to improve the energy balance from the perspectives of genetics, nutrition and bio-pharmaceuticals. low rfi selection made dramatic changes in some biological pathways possible. for example, the cell death and apoptosis of tumor cell lines, activation of antigen presenting cells and cell viability of kidney cell lines were decreased during low rfi selection. additionally, chickens with low rfi were more prone to vascular lesions. every change was a result of the mutual action of degs.table predictable changes in biological functioning based on deg analysis in low-rfi individuals apoptosis and apc viability warranted attention because they were tightly linked to immunity. apoptosis was effectively under the control of genes. when low-rfi birds suffer from avian leukosis virus, marek’s disease or reticuloendotheliosis, related degs might be responsible for blocking the energy supply to tumor cells, thereby slowing apoptosis and accelerating tumor formation. apcs are capable of ingesting and processing pathogenic microorganisms by phagocytosis or pinocytosis, which in turn produces peptide fragments that contain antigenicity domains. decreased activation of apcs in low-rfi birds might cause a delay in the immune response or a reduced ability to kill pathogens. hsp90b is an important immune protein that is involved in cell protection under heat stress. increased hsp90b expression in response to heat stress causes various reductions in cell damage and even repairs damaged proteins. the reduced hsp90b expression in low-rfi birds signified a weaker ability to resist the effect of environmental factors compared to high-rfi birds. additionally, the changes in some degs were found to function in the regulation of fat metabolism. for low-rfi birds, degs were predicted to facilitate the concentration of lipids, were predicted to play a pivotal role in the activation of lipid synthesis, and were associated with the activation of fatty acid metabolism. although a few degs had inhibitory effects, the above three specific physiological functions seemed to be enhanced overall. the results in the present study showed that low-rfi birds could achieve greater synthetic metabolism of lipids compared to high-rfi birds, whereas the results of another study showed that they displayed weaker catabolic abilities. the faster anabolism of fat than catabolism in low-rfi birds might cause abnormal storage of fat and a decrease in heat release.fig. predictable changes in fat metabolism based on deg analysis in low-rfi individuals among the lipometabolism-related degs, pck was particularly noteworthy because it was remarkably increased in the low-rfi group. the pck gene is associated with obesity, insulin resistance, and type ii diabetes in mammals. abdominal fat content was assumed to be positively correlated with pck mrna expression in birds. thus, high pck expression in low-rfi birds might signal a concurrent increase in the abdominal fat mass. interestingly, ar expression was significantly lower in the low-rfi birds than in the high-rfi birds. the interaction of androgen with its receptor was closely related to bone metabolism in birds. the ar in the cytoplasm enters the nucleus by integrating with androgen and then binding to the androgen responsive element, leading to the activation of androgen. low ar expression interferes with the positive physiological effect of androgen, leading to high pck expression. a systematic analysis of the gwas and rna sequencing results was performed. the results showed there was no overlap of the genes within the 50 kb flanking region containing the significant snps detected by gwas and any deg detected by rna sequencing. one explanation is that the actual linkage distance between the qtls and the significant snp is larger than 50 kb. however, we stopped measuring the rfi when the chickens were d of age, whereas the liver sample collection was performed at d of age. during this time, the rfi ranking may have slightly changed, resulting in a mismatch. nonetheless, when the search scope was broadened we still found that some potential genes within the 500-kb flanking region of the significant rfi-related snps in gwas were identified as degs in chickens with divergent rfis, including mgp, hist1h hist1h2a4l and oc near two snps in gga nr0b near the snp rs per near the snp rs st6galnac near the snp rs and g0s near the snp rs notably, the concurrent genes involved in gwas and rna sequencing played critical roles in a wide spectrum of biological processes. therefore, their relationships with rfi need to be verified.  the heritability of rfi during the period from to d of age was moderate. the rfi was particularly affected by significant snps that could explain  % of the additive genetic variance. seventeen rfi-related snps were located in a 1 mb region of gga this region was identified as a key candidate region affecting the energy utilization efficiency of chickens because it contained genes associated with lipogenesis, social behavior, and immunity. although they obtained less metabolic energy, low-rfi birds maintained normal growth that was comparable to that of the high-rfi birds by allocating less energy to cell activity, the immune response, and physical activity in relation to eating and thus optimizing the use of the limited resources. nevertheless, low-rfi selection cannot mobilize spare energy to withstand environmental stress and causes potential health risks that should not be ignored. at the same time, the low-rfi birds tended to become fatter. 
similar results were obtained in the transcriptome sequencing experiments. a lower average daily feed intake was found in low-rfi birds compared to high-rfi birds. some of these genes were relevant to appetite, cell activities, and fat metabolism, such as cckar, hsp90b and pck some potential genes within the kb flanking region of the significant rfi-related snps detected in gwas were also identified as degs in chickens with divergent rfis. low rfi selection enables a reduction in feed intake without affecting growth performance. however, the effective variants or major genes dedicated to phenotypic differences in rfi in quality chickens are unclear. residual feed intakegenome-wide association studyrna-sequencingquality chickensthe high technology research and development program of china2013aa102501zhang xiquan the national key technology research and development program2014bad08b08zhang dexiang the china agriculture research systemcars-42-g cars-42-z17zhang xiquan issue-copyright-statement© the author 2016 more than half of the effective snps were located in a 1 mb region of chicken chromosome thus, focusing on this region should enable a deeper understanding of energy utilization. the gwas findings showed that the 1 mb narrow region of gga should be important because it contained genes involved in energy-consuming processes, such as lipogenesis, social behavior, and immunity. residual feed intake is a powerful indicator for energy utilization efficiency and responds to selection. in general, low-rfi birds seemed to optimize energy employment by reducing energy expenditure in cell activities, immune responses, and physical activity compared to eating. rna sequencing was performed to profile the liver transcriptomes of four male chickens selected from the high and low tails of the rfi. gwas showed that of the significant single nucleotide polymorphisms associated with the rfi accounted for  % of the additive genetic variance.
 residual feed intake is a powerful indicator for energy utilization efficiency and responds to selection. low rfi selection enables a reduction in feed intake without affecting growth performance. however, the effective variants or major genes dedicated to phenotypic differences in rfi in quality chickens are unclear. therefore, a genome-wide association study and rna sequencing were performed on rfi to identify genetic variants and potential candidate genes associated with energy improvement.  a lower average daily feed intake was found in low-rfi birds compared to high-rfi birds. the heritability of rfi measured from to d of age was. gwas showed that of the significant single nucleotide polymorphisms associated with the rfi accounted for  % of the additive genetic variance. more than half of the effective snps were located in a 1 mb region of chicken chromosome thus, focusing on this region should enable a deeper understanding of energy utilization. rna sequencing was performed to profile the liver transcriptomes of four male chickens selected from the high and low tails of the rfi. one hundred and sixteen unique genes were identified as differentially expressed genes. some of these genes were relevant to appetite, cell activities, and fat metabolism, such as cckar, hsp90b and pck some potential genes within the kb flanking region of the significant rfi-related snps detected in gwas were also identified as degs in chickens with divergent rfis.  the gwas findings showed that the 1 mb narrow region of gga should be important because it contained genes involved in energy-consuming processes, such as lipogenesis, social behavior, and immunity. similar results were obtained in the transcriptome sequencing experiments. in general, low-rfi birds seemed to optimize energy employment by reducing energy expenditure in cell activities, immune responses, and physical activity compared to eating. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords residual feed intakegenome-wide association studyrna-sequencingquality chickensthe high technology research and development program of china2013aa102501zhang xiquan the national key technology research and development program2014bad08b08zhang dexiang the china agriculture research systemcars-42-g cars-42-z17zhang xiquan issue-copyright-statement© the author 2016 