small synthetic molecules termed growth hormone secretagogues ( ghss ) act on the pituitary gland and the hypothalamus to regulate growth hormone ( gh ) release . the growth hormone secretagogue receptor ( ghsr or ghs - r ) , a g protein - coupled receptor , has been cloned from human and pig and is mainly expressed in the pituitary , hypothalamus , and hippocampus . in 1999 , a novel 28-amino acid gut - brain peptide named ghrelin ( ghrl ) was first isolated from the mammalian stomach . similar to the synthetic molecules of ghss , ghrl proved to be an endogenous ligand for ghsr and involved in gh secretion , food intake , and energy homeostasis [ 2 , 3 ] . even though the mature ghrl varies in structure among mammals , birds , and fish , the n - octanoylation of the ser-3 residue it is now well known that ghrl interacts with ghsr to mediate a variety of bioactivities through the neuroendocrine pathway . body mass index and body fat were shown to be negatively correlated with plasma ghrl levels [ 58 ] . two polymorphisms , arg51gln and leu72met , in the human ghrl gene were identified in obese subjects , and leu72met was significantly associated with body mass index and fat mass in some human populations [ 9 , 10 ] . in addition to these two nonsynonymous snps , two other variations of gln90leu and a frameshift mutation ( 2  bp deletion at codon 34 ) were also reported in the human ghrl gene , and the 90leu allele frequency was significantly higher in extremely obese children and adolescents ( 0.063 ) than in normal - weight students . a novel snp , t3056c in intron 2 of the ghrl gene , was significantly associated with the acylation of ghrl and some fatness traits , including high body mass index , body weight , fat mass , and skinfold thickness . among 11 snps identified in the 5 flanking region of the ghrl gene , snp-501a > c was significantly associated with body mass index in human . in mice , the peripheral administration of ghrl was found to regulate body weight , adiposity , and ucp mrna expression . until now , the research progress on the ghrl and ghsr genes was achieved in chicken . the chicken ghrl ( cghrl ) gene cdna was first cloned by kaiya et al . , and it encoded a 116-amino acid ghrl precursor and a 26-amino acid mature ghrl . the first exon did not encode any amino acids , a characteristic that was conserved in mammals [ 17 , 18 ] . the cghsr gene was composed of two exons separated by an intron and encoded a normal ghsr with seven transmembrane domains ( tm1 to tm7 ) ; however , a variant ghsr without tm6 due to a 48-bp deletion was found . a total of 19 and 37 snps were identified in the cghrl and cghsr genes , respectively , and an 8-bp indel in the 5 untranslated region ( utr ) and some snps of the cghrl gene were related to chicken growth and muscle fiber traits [ 2123 ] . the duck ghrl ( dghrl ) gene cdna was also cloned , and the encoded dghrl was highly similar to cghrl . nevertheless , no further studies on the dghrl and duck ghsr ( dghsr ) genes have been reported . most fat accumulates as abdominal fat in chicken but as subcutaneous fat in duck ; these characteristics make the chicken and duck ideal for use in this study . race , genome walking , real time pcr , and association analysis were performed to characterize the ghrl and ghsr genes and reveal their effects on fat deposition in chicken and duck . a total of 411 birds ( 219 males and 193 females ) from 8 chicken populations ( pop1 to pop8 ) and 139 birds from 9 duck populations ( pop9 to pop17 ) were used in this study ( table 1 ) . these samples consisted of six chicken breeds , including xinghua ( xh ) , white recessive rock ( wrr ) , taihe silkies ( ts ) , leghorn ( lh ) , lingnan yellow ( ly ) , huiyang beard ( hb ) , and an f2 full - sib hybrid population ( xh&wrr ) described by fang et al . , as well as four duck breeds , sanshui white ( sw ) , peking ( pk ) , partridge ( pt ) , and lake ( lk ) . either blood or live tissues were collected from these birds at 9 weeks of age for the purpose of sequence cloning , variation , mrna quantitative expression , cell culture , and marker trait association analyses ( table 1 ) . the sw commercial population ( pop14 ) , including 100 ducks ( 52 males and 48 females ) , were slaughtered at 90 days old , and body weights at hatch ( g ) and 90 days old ( g ) , carcass weight ( g ) , fat thickness under skin ( mm ) , fat width ( mm ) , eviscerated weight ( g ) , abdominal fat pad weight ( g ) , and abdominal fat pad ratio ( % ) were recorded . all birds were raised in separate pens and fed with commercial corn - soybean - based diets that met nrc requirements . ( guangdong , china ) , and all ducks were provided by guangdong sanshui lianke duck farm ( guangdong , china ) . total rna was extracted from the isolated tissues using an improved trizol isolation method ( invitrogen , california , usa ) following the manufacturer 's instructions . one microgram of total rna was used to obtain cdna by reverse transcription with use of the rever tra ace transcriptase kit ( toyobo , japan ) and random hexamers . thirty - three primer pairs ( pm1 to pm33 ) were designed and consisted of pm1 to pm6 ( 6 ) for the cghrl gene , pm7 to pm10 ( 4 ) for the cghsr gene , pm11 for the chicken -actin gene , pm12 to pm24 ( 13 ) for the dghrl gene , pm25 to pm32 ( 8) for the dghsr gene , and pm33 for the duck -actin gene ( table 2 ) . pm12 and pm26 were both genomic primers , each of which consisting of three reverse primers and a random forward primer supplied by the genomic walking kit . pm14 was a primer pair for 3 race , and the remaining 30 primer pairs were gene - specific primers . the genetool software ( http://www.biologysoft.com/ ) was used to design the primers based on reported sequences of the chicken ghrl ( genbank ab075215 ) , ghsr ( genbank ab095994 ) , and -actin genes ( genbank nm_205518 ) , as well as duck ghrl ( genbank ay338466 ) , ghsr ( genbank eu005225 ) , -actin ( genbank ef667345 ) , and goose ghrl ( genbank ay338465 ) . pcr was performed in 25  l reactions containing 50  ng chicken genomic dna , 1   pcr buffer , 12.5  pmol primers , 100  m of each dntp , 1.5  mm mgcl2 , and 1.0  u taq dna polymerase ( sangon biological engineering technology company , shanghai , china ) . usa ) with the following procedure : 3 minutes at 94c ,   followed by 35 cycles of 30 seconds at 94c ,   45 seconds at the annealing temperature ( 5862c ) and 1 minute at 72c , and a final extension of 5 minutes at 72c . the pcr product of pm5 was sequenced for genotyping of the a-2220c , a-2246 g , a-2355c , c-2399del the pcr product of the pm7 reaction was digested with tat i with the pcr - rflp method for genotyping c-1896a in the cghsr gene . the pcr product of the pm8 reaction was digested by hin1 ii with the pcr - rflp method for genotyping the c-1459 t snp in the cghsr gene . the pcr product of the pm9 reaction was digested with tail i with the pcr - rflp method for genotyping the a-1022c snp in the cghsr gene . the pcr product of the pm20 reaction was digested with csp6 i with the pcr - rflp method for genotyping the c-729 t snp in the dghrl gene . the pcr product of the pm22 reaction was digested with tail i with the pcr - rflp method for genotyping the t985c snp in the dghrl gene . the pcr product of the pm30 reaction was digested with csp6 i with the pcr - rflp method for genotyping the t404c snp in the dghsr gene . the pcr product of the pm31 reaction was digested with csp6 i with the pcr - rflp method for genotyping the a3427 g snp in the dghsr gene . digestion products were detected by agarose gel ( 1.5% ) electrophoresis , and genotypes were determined by specific profiles ( supplementary figure 1 in supplementary material available on - line at doi : 10.1155/2009/567120 ) . total rna was extracted from the proventriculus from an sw duck by the trizol reagent ( invitrogen , carlsbad , ca ) following the manufacturer 's instructions . a proventriculus cdna library was prepared using the smart race cdna amplification kit ( clontech laboratories , mountain view , ca ) . primer - directed rt - pcr and 3 rapid amplification of cdna ends ( race ) were used to generate duck ghrl cdnas , amplified using primers p1 to p3 ( table 2 ) . a cdna library was prepared using the smart race cdna amplification kit ( clontech laboratories , mountain view , ca ) . the 3 ends of the cdnas were amplified with a specific forward primer and reverse random primers from the smart race cdna amplification kit following the manufacturer 's instructions . to obtain the 5 flanking region of the duck ghrl and ghsr genes , the duck genomic dna was purified using the genomic dna purification kit ( u - gene , china ) and was then used for genome walking . the genome walking procedure included three reactions : the first pcr reaction used the outer adaptor primer ap1 from the kit and an outer gene - specific primer pm12r1 ( or pm26r1 ) ; the second reaction used primers ap2 and pm26r2 ; the third reaction used primers ap3 and pm26r3 . pcr products of the third reaction were purified using the gel extraction kit ( u - gene , china ) , cloned into the pgem - t easy vector ( promega , usa ) , and sequenced by shanghai biosune biotechnology co. ltd . the mrna levels of ghrl and ghsr in 18 tissues of chickens and ducks were determined by quantitative real time pcr with sybr green dye using -actin as an internal positive control ( ipc ) . the 15  l pcr mixture contained 1  l of cdna , 7.5  m super mix ( toyobo , japan ) , 10  m of each primer ( pm6 , pm10 , pm11 , pm24 , pm32 , and pm33 ) , and 6.0  l depc h2o . real time pcr was run on an abi 7500 ( applied biosystems , foster city , ca , usa ) using the following program : initial denaturation at 95c   for 4 minutes , then 40 cycles of 94c for 30 seconds , 63c for 30 seconds , and 72c for 45 seconds . each sample was repeated three times and the average mrna level was obtained for further analysis . two random pcr products of pm6 , pm10 , pm11 , pm24 , pm32 , and pm33 were sequenced by shanghai biosune biotechnology co. ltd . ( shanghai , china ) to confirm that specific fragments of the cghrl , cghsr , chicken -actin , dghrl , dghsr , and duck -actin genes were obtained . quantitative values were obtained from the ct values , which were the inverse ratio relative to the starting pcr product . the relative quantification was obtained by 2 in which ct = cttarget gene  ctipc . statistics were analyzed by sas students t - test that measures analysis of variance with a significance level of 0.05 . the homology of ghrl among human , mouse , chicken , turkey , emu , goose , and duck was studied . identity percentages were obtained from amino acid blast with the dnastar software ( http://www.dnastar.com/ ) . the obtained sequences of ghrl and ghsr were aligned with the mega 3.1 software ( http://www.megasoftware.net/ ) . the 798-bp 5 flanking region of the dghrl gene obtained in this study was used to predict the potential binding sites for transcription factors by an online service ( http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl ) . in the chicken f2 population ( xh&wrr ) and the duck commercial population ( sw ) , haplotypes were inferred based on the genotype data with the phase 2.1 software ( http://en.wikipedia.org/wiki/phase ) . to evaluate the nucleotide diversity , the normalized numbers of snps (  ) were calculated by dividing k ,   the number of observed nucleotide changes , by the total sequence length in base pairs ( l ) and correcting for the sample size ( n ) as described in , using the following formula : 
										 ( 1)=ki=1n1i1l . the effects of 13 snps identified in this study on fatty traits were estimated by marker - trait association analysis . these snps included six in the cghrl gene ( c-2030 t , c-2047 g , a-2220c , a-2246 g , a-2355c , and c-2399del ) , three in the cghsr gene ( c-1896a , c-1459 t , and a-1022c ) , two in the dghrl gene ( c-729 t and t985c ) , and two in the dghsr gene ( t404c and a3427 g ) . marker - trait association analysis was performed using the sas glm procedure ( sas institute , 1996 ) and the genetic effects were analyzed by a mixed procedure according to the following model : 
 ( 2)y=+g+d+h+s+e , 
									 where y is an observation on the trait ,  is the overall population mean , g is the fixed effect of the genotype , d is the random effect of dam , h is the fixed effect of hatch , s is the fixed effect of sex ( male or female ) , and e is the residual random error . the full - length cdna of the dghrl gene ( 835  bp ) was amplified using primers pm13 to pm15 . it comprised 115  bp of the 5 untranslated region ( 5 utr ) , the 351  bp open reading frame ( orf ) encoding a 116-amino acid ( aa ) peptide , and 369  bp of the 3 utr ( figure 1 ) ( genbank ef613551 ) . the sw duck ghrl precursor ( 116 aa ) included a signal peptide ( 23 aa ) , the mature ghrl ( 26 aa ) , and a long c - terminal peptide ( 67 aa ) . compared with the other reported mallard dghrl precursor sequence ( ncbi accession number : ay338466 ) , there were some different amino acids ( w16r , g21a , a53 t , t59a , a64 t , and g71s ) . the putative   sw dghrl precursor showed identities of 88.8% , 72.4% , 69.8% , 71.6% , 37.9% , and 37.1% with its counterparts of goose , chicken , turkey , emu , mouse , and human , respectively ( figure 2 ) . as far as the mature peptides are concerned , however , dghrl had much higher homology with the other six species ( 92.3% , 65.4% , 65.4% , 69.2% , 53.8% , and 50.0% with goose , chicken , turkey , emu , mouse , and human , resp . ) ( figure 2 ) . the sequence ensemble generated with the pm12 to pm19 products gave rise to a 3670  bp genomic fragment of the dghrl gene , which included 798  bp of the 5 flanking region , 883  bp of five exons ( 134 , 137 , 114 , 109 , and 389  bp for exon 1 to exon 5 ) and 1989  bp of four introns ( 168 , 469 , 458 , and 894  bp for intron 1 to intron 4 ) . like other species , the four introns of the dghrl gene all followed the  gt - ag rule , and moreover , exon 1 did not encode any amino acids ( supplementary figure  2 ) . the obtained dghrl genomic sequences were submitted to the ncbi database ( genbank ef613552 ) . the 3717  bp sequence of the dghsr gene was deduced by genomic walking ( pm26 ) , pcr amplification ( pm25 ) and sequence ensemble with reported sequences ( genbank eu005225 ) . it comprised two incomplete exons ( 591 and 148  bp for exon 1 and exon 2 ) and one intron ( 2978  bp ) that followed the  gt - ag  intron rule ( supplementary figure  2 ) . the obtained dghsr gene sequences were also submitted to the ncbi database ( genbank fj194548 ) . online prediction of transcription factor binding sites in the 5 flanking region of the dghrl gene showed that a typical tata box was present in 23  bp upstream of the transcription start site , and potential binding sites for some transcription factors were found , including one aml-1a site ( runt - factor aml-1 ) , two cap 1 sites ( cap signal for transcription initiation ) , two cdxa 1 sites , three gata-1 sites ( gata - binding factor 1 ) , as well as one oct-1 site ( octamer factor 1 ) ( figure 3 ) . a total of 19  snps ( 10 transitions , 8 transversions , and 1 indel ) and 1 polya polymorphism were identified in 2751  bp of the 5 utr region of the cghrl gene , while 8 snps ( 5 transitions and 3 transversions ) were identified in 1358  bp of the 5 utr of the cghsr gene ( table 3 ) . forty - three snps ( 27 transitions , 15 transversions , and 1 triallelic snp ) , 1 ssr , and 1 9-bp indel were identified in 3671  bp of the dghrl gene , while 48 snps ( 35 transitions and 13 transversions ) and 5 indels were found in 3567  bp of the dghsr gene ( table 3 ) . eleven snps were located in the coding regions of the dghrl gene , but only four snps were nonsynonymous and led to amino acid changes in the c - terminal extension peptide . for the dghsr gene , however , no nonsynonymous snps were found within five snps in the coding regions ( table 3 ) . a triallelic snp ( ga / c at nt 1774 ) was observed in the coding region of the dghrl gene , which was related to three different amino acids ( a / p / t at aa 59 ) ( table 3 ) . the average bps per snp were 145 , 170 , 86 , and 75 for the cghrl , cghsr , dghrl , and dghsr genes , respectively . the nucleotide diversities (  ) corrected for sample size were 1.83  10 , 1.56  10 , 3.10  10 , and 3.56  10 for the cghrl , cghsr , dghrl , and dghsr genes , respectively . the highest mrna level of the cghrl gene was found in the proventriculus and then the pituitary , cerebrum , abdominal fat , subcutaneous fat , hypothalamus , small intestine , heart , and breast muscle . the highest mrna level of the dghrl gene was also found in the proventriculus and then the abdominal fat , testicles , subcutaneous fat , uropygial gland , and breast muscle ( figure 4 ; supplementary table 1 ) . the highest mrna level of the cghsr gene was detected in the breast muscle and then the subcutaneous fat , leg muscle , abdominal fat , heart , spleen , liver , uropygial gland , cerebrum , proventriculus , pituitary , and testicles . the highest mrna level of the dghsr gene was detected in the pituitary and then the subcutaneous fat , hypothalamus , small intestine , testis , cerebellum , and cerebrum , whereas little mrna was found in the other tissues ( figure 4 ; supplementary table 1 ) . when the hb and ly chicken breeds were compared , significant differences in the ghrl mrna levels were found in the proventriculus , subcutaneous fat , lung , and kidney ( p < .05 ) , whereas significant differences in the ghsr mrna levels were found in the hypothalamus and breast muscle ( p < .05 ) . when the pt duck and sw duck breeds were compared , significant differences in the ghrl mrna levels were found in the proventriculus and subcutaneous fat ( p < .05 ) , whereas no significant differences in the ghsr mrna levels were found in any tissues ( p > .05 ) ( figure 4 ; supplementary table  1 ) . for the cghrl gene , c-2047 g and a-2355c were found to be significantly associated with abdominal fat weight ( afw ; p = .01 < .05 ) and crude protein content of leg muscle ( cpclm ; p = .02 < .05 ) , and a-2220c was significantly associated with cpclm ( p = .0009 < .01 ) . in addition , haplotypes across six snps ( c-2030 t , c-2047 g , a-2220c , a-2246 g , a-2355c , and c-2399del ) were significantly associated with afw ( p = .04 < .05 ) , abdominal fat ratio ( afr ; p = .04 < .05 ) , and cpclm ( p < .0001 ) . as far as the three genotypes of each locus were concerned , cc of c-2047 g , cc of a-2220c , and aa of a-2355c had the highest values of association with afw and cpclm , indicating that the advantageous alleles for fat deposition were c-2047 , a-2220 , and a-2355 ( table 4 ) . for the cghsr gene , c-1459 t was shown to be significantly associated with cpclm ( p = .0004 < .01 ) . individuals with the cc genotype had the highest cpclm association , indicating that c rather than t was the dominant allele for cpclm ( table 4 ) . in duck , c-729 t and haplotypes of c-729 t and t985c of the dghrl gene were both significantly associated with subcutaneous fat thickness ( sft ; p = .04 and .05 , resp . ) . individuals with the ct genotype of c-729 t had the highest sft . for the dghsr gene , a3427 g and haplotypes of t404c and a3427 g were both significantly associated with sft ( p = .04 and .05 , resp . ) . individuals with the tt genotype of c3427 t had the highest sft , indicating that t rather than c was the dominant allele for fat deposition ( table 4 ) . the genomic organization of the ghrl and ghsr genes was conserved in chicken and duck ( supplementary figure  2 ) . the cdnas of the cghrl and cghsr genes were reported by kaiya et al .   and tanaka et al . the reported cghrl and cghsr genes were 2706 and 4121  bp long , respectively , and comprised five and two exons each [ 17 , 19 , 27 ] . from the cloning and sequencing performed in this study , the dghrl cdna ( genbank ef613551 ) had a 351-bp orf that encodes a 116-aa precursor . the predicted mature dghrl was a 26-aa peptide , and only two amino acids were different from cghrl . the first seven amino acids , including a serine residue at position 3 ( site of n - octanoylation ) , of the mature dghrl are identical to those of chicken , turkey , goose , and emu ( figure 2 ) [ 17 , 24 ] . the obtained dghrl gene sequence ( genbank ef613552 ) also comprised five exons and four introns , and the first exon did not encode any amino acids . this characteristic was conserved in chicken , turkey , human , and rodent [ 1618 , 28 , 29 ] . the obtained 3717 bp sequence of the dghsr gene ( genbank fj194548 ) consisted of two exons and one intron and encoded a 347-aa mature receptor protein . all introns of the cghrl , cghsr , dghrl , and dghsr genes followed the gt - ag rule , which is conserved in eukaryotic genes . online prediction indicated that a typical tata box and a number of transcriptional factor binding sites ( aml-1a , cap 1 , cdxa 1 , gata-1 , and oct-1 ) were present in the 5 flanking region of the dghrl gene . these transcriptional factor binding sites were also found in the ghrl gene promoter regions of human and chicken [ 29 , 30 ] , and a  tatataa  sequence was reported to be located at the cghrl gene promoter . gata regulatory motifs were first found in the promoters of globulin and other erythroid - specific genes . aml-1 is also a key regulator of hematopoiesis and plays an important role in development of all hematopoietic lineages . it is possible that dghrl gene expression in hematopoietic cells would be regulated by gata-1 and aml-1a . the transcription factor cap-1 is a key factor of the cap - dependent translation initiation mechanism . oct-1 is one of the transcription factors with a pou homeodomain and is expressed in most tissues including the brain . moreover , oct-1 plays very important roles in the transcriptional regulation of gonadotropin - releasing hormone and aldolase c gene expression in the brain [ 34 , 35 ] . cdxa is one of the vertebrate caudal proteins that play important roles in establishment of the body plan during early development . these transcription factor binding sites in the putative promoter region might affect dghrl gene expression . the ghrl and ghsr genes were found to be more variable in poultry than in mammals . as indicated by this study , the snp densities were 145 , 170 , 86 , and 75  bp per snp for the cghrl ( 2751  bp/19 snp ) , cghsr ( 1358/8 ) , dghrl ( 3671/43 ) , and dghsr ( 3567/48 ) genes , respectively ( table 3 ) . in humans , a total of 11 snps were found in 1657  bp of ghrl gene with an snp density of 1 snp/151  bp . the reported density for the human ghsr gene ( 5 snp of 1101  bp ) was 220  bp per snp . based on the dbsnp data ( http://www.ncbi.nlm.nih.gov ) , a total of 8 and 32 snps were reported in 2922 and 6494  bp of the mouse ghrl and ghsr genes , with estimated densities of 365 and 203  bp per snp , respectively . it was indicated that the higher variations of the ghrl and ghsr genes were found in chicken and duck compared with those of human and mouse . moreover , as far as these two species were concerned , higher snp densities were found in duck compared to chicken , as the nucleotide diversity of the dghrl ( 3.10  10 ) and dghsr ( 3.56  10 ) genes were much higher than those of cghrl ( 1.83  10 ) and cghsr ( 1.56  10 ) . our recent study on the chicken and duck thrsp    genes also showed higher variations in the duck genome ( data not shown ) . as indicated in this study , the ghrl and ghsr genes were associated with fatness traits in chicken and duck , which is consistent with some previous studies in humans . many studies showed associations of the ghrl gene with adiposity [ 1012 , 38 ] and type 2 diabetes   in humans . additionally , some polymorphisms in the ghsr gene were found to be associated with obesity , bulimia nervosa , and pharmacological abnormalities in humans [ 4043 ] . in chickens , the association of cghrl polymorphisms with growth traits has been reported [ 22 , 23 ] . moreover , significant association of the cghsr gene with several fatness traits was reported by a recent study . in this study , the ghrl and ghsr genes were found to be associated with several fatness traits like afw , cpclm , and sft in both chicken and duck . analysis of mrna levels also showed the effects of the ghrl and ghsr genes on fat deposition in poultry . even though the ghrl gene was predominantly expressed in the proventriculus , high mrna levels of the ghrl and ghsr genes were detected in subcutaneous fat and abdominal fat in both chicken and duck ( figure 4 ) . this suggests that both ghrl and ghsr genes may be involved in fat mobilization in poultry . moreover , much higher ghrl mrna levels were found in ly ( 2.07  0.06 ) rather than hb ( 0.07  0.05 ) chickens ; higher levels were found in sw ( 0.78  0.01 ) compared to those in pt ( 0.13  0.01 ) ducks ( supplementary table  1 ) . sw ducks and ly chickens are two fast - growing commercial breeds with higher fat contents compared with those of the other two native breeds of pt ducks and hb chickens . the higher ghrl mrna in higher - fat breeds suggested that ghrl was probably advantageous for fat deposition in poultry . from these investigations , the variations and expression patterns of the cghrl , cghsr , dghrl , and dghsr genes were characterized , and some snps of these genes were related to fatty traits .
ghrelin ( ghrl ) and its receptor ( ghsr ) are involved in various bioactivities . in this study , 
 the complete cdna and 5 flanking region of the duck ghrl ( dghrl ) gene and a 3717  bp fragment of the duck ghsr ( dghsr ) gene were obtained . a total of 19 , 8 , 43 , and 48  snps identified in 2751 , 1358 , 3671 , and 3567  bp of the chicken ghrl ( cghrl ) , chicken ghsr ( cghsr ) , dghrl , and dghsr genes , respectively . 
 both cghrl and dghrl were expressed predominantly in the proventriculus , whereas the highest mrna levels of cghsr and dghsr were detected in the breast muscle and pituitary . 
 association analysis showed that c-2047 g , a-2355c , and a-2220c of the cghrl gene were significantly associated with abdominal fat weight ( afw ; p = .01 ) , crude protein content of leg muscle ( cpclm ; p = .02 ) , and cpclm ( p = .0009 ) , respectively . 
 c-1459 t of the cghsr gene was also significantly associated with cpclm ( p = .0004 ) . 
 c-729 t of dghrl and   a3427 t of dghsr were both significantly associated with subcutaneous fat thickness ( sft ; p = .04 ) . 
 it was indicated by this study that the ghrl and ghsr genes were related to fat deposition in both chicken and duck .