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Laying records on 1,534 F2 hens, derived from a reciprocal cross between White Leghorns and Dongxiang blue-shelled chickens, were used to estimate genetic parameters for residual feed intake (RFI), feed conversion ratio (FCR), daily feed intake (FI), metabolic BW (MBW), BW gain (BWG), and daily egg mass (EM) at 37 to 40 (T1) and 57 to 60 wk age (T2), respectively. Genetic analysis was subsequently conducted with the AI-REML method using an animal model. Estimates for heritability of RFI, FCR, and FI were 0.21, 0.19, and 0.20 in T1, and 0.29, 0.13, and 0.26 in T2, respectively. In T1 and T2, RFI showed high and positive genetic correlations with FCR (0.51, 0.43) and FI (0.72, 0.84), whereas the genetic correlation between FI and FCR was very low (-0.09, 0.11). Genetically, negative correlations were found between RFI and its component traits (-0.01 to -0.47). In addition, high genetic correlations, from 0.76 to 0.94, were observed between T1 and T2 for RFI, FCR, and FI, suggesting that feed efficiency traits in the 2 stages had a similar genetic background. The results indicate that selection for low RFI could reduce FI without significant changes in EM, while selection on FCR will increase EM. The present study lays the foundation for genetic improvement of feed efficiency during the laying period of chickens.

Endogenous retroviruses are regarded as ideal genetic markers for evolutionary analyses. Birds were some of the initial vertebrates found to contain endogenous retroviruses. However, few studies have investigated the presence and distribution of endogenous retroviruses in goose. In this study, we detected the avian sarcoma and leukosis virus gag gene in the genomic DNA of 8 Chinese native breeds using polymerase chain reaction method. The results indicated that a 1.2-kb avian sarcoma and leukosis virus gag sequence was integrated into all 8 goose breeds. The mean genetic pairwise distance was 0.918% among the investigated geese. To the best of our knowledge, this is the first report demonstrating the presence of the endogenous retroviruses in the domestic goose genome. The genetic structure should be further examined in the domestic goose.

Beijing-you is a Chinese local chicken which is raised for both meat and eggs. In the present study, we detected the effects of different rearing systems on growth, slaughtering performances and meat quality of Beijing-you chickens at 26-40 weeks of age. Six hundred Beijing-you hens were randomly allocated into two groups at 16 weeks of age and raised in free range or battery cage systems. The body weight, slaughtering performance and meat quality were measured for each group at the ages of 26, 30, 35 and 40 weeks. Some of the traits were dramatically influenced by the two systems, although most of them did not show significant changes. For the meat fiber microstructure, we found that the diameter of thigh and breast muscle fiber in the free range group were significantly increased than in the cage group (P < 0.05) at 26 weeks of age. The ratio of fast muscle fiber in thigh muscle samples of the free range group was significantly reduced compared to that of cage group at both 35 (P < 0.01) and 40 (P < 0.01) weeks of age, indicating that the free range system could promote the transforming of fast muscle fiber to slow muscle fiber.

Shank color of domestic chickens varies from black to blue, green, yellow, or white, which is controlled by the combination of melanin and xanthophylls in dermis and epidermis. Dermal shank pigmentation of chickens is determined by sex-linked inhibitor of dermal melanin (Id), which is located on the distal end of the long arm of Z chromosome, through controlling dermal melanin pigmentation. Although previous studies have focused on the identification of Id and the linear relationship with barring and recessive white skin, no causal mutations have yet been identified in relation to the mutant dermal pigment inhibiting allele at the Id locus. In this study, we first used the 600K Affymetrix Axiom HD genotyping array, which includes ~580,961 SNP of which 26,642 SNP were on the Z chromosome to perform a genome-wide association study on pure lines of 19 Tibetan hens with dermal pigmentation shank and 21 Tibetan hens with yellow shank to refine the Id location. Association analysis was conducted by the PLINK software using the standard chi-squared test, and then Bonferroni correction was used to adjust multiple testing. The genome-wide study revealed that 3 SNP located at 78.5 to 79.2 Mb on the Z chromosome in the current assembly of chicken genome (galGal4) were significantly associated with dermal shank pigmentation of chickens, but none of them were located in known genes. The interval we refined was partly converged with previous results, suggesting that the Id gene is in or near our refined genome region. However, the genomic context of this region was complex. There were only 15 SNP markers developed by the genotyping array within the interval region, in which only 1 SNP marker passed quality control. Additionally, there were about 5.8-Mb gaps on both sides of the refined interval. The follow-up replication studies may be needed to further confirm the functional significance for these newly identified SNP.

Resistance to diseases varies considerably among populations of the same species and can be ascribed to both genetic and environmental factors. Salmonella Pullorum (SP) is responsible for significant losses in the poultry industry, especially in developing countries. To better understand SP resistance in chicken populations with different genetic backgrounds, we orally challenged 3 chicken lines with SP-a highly selected commercial breed (Rhode Island Red, RIR), a local Chinese chicken (Beijing You, BY), and a synthetic layer line (dwarf, DW)-at 4 d of age. Two traits related to SP resistance, survival, and bacterial carriage in the spleen were evaluated after infection. Survival rates were recorded up to 40 d of age when all chickens still alive were killed to verify the presence of SP in the spleen to determine carrier state. Mortalities for RIR, BY, and DW chicks were 25.1%, 8.3%, and 22.7%, respectively, and the corresponding carrier-states in the spleens were 17.9%, 0.6%, and 15.8%. Survival and carrier-state heritabilities were estimated using an animal threshold model. Survival heritability was 0.197, 0.091, and 0.167 in RIR, BY, and DW populations, respectively, and the heritabilities of carrier state for DW and RIR were 0.32 and 0.16, respectively. This is the first time that the heritability of the SP carrier state has been evaluated in chickens. Our study provides experimental evidence that chickens with various genetic background exhibited significantly different SP-resistant activities and heritabilities. These results may be useful for selecting lines with better disease resistance.

Flavin-containing monooxygenase 3 (FMO3) is an important monooxygenase for catalytic oxygenation of many harmful xenobiotics. Mutations in the FMO3 gene have been identified as causing trimethylaminuria in human and fishy off-flavor in cow milk and chicken eggs. In this study, the full-length cDNA sequence of Pekin duck FMO3 gene was cloned, sequenced, and characterized. The full-length cDNA sequence consisted of 1,846 bp and contained a 1,599 bp open-reading frame encoding 532 amino acids. Duck FMO3 gene shared a similar nine exon-eight intron structure with chicken and human. The duck FMO3 putative protein sequence showed high identity with that of chicken (82 %), and relative low identity with those of mammals (61-66 %). We also found that the duck FMO3 gene was dramatically expressed in liver, lung, and kidney compared to that in other tissues in the ducks, indicating the possible roles the FMO3 gene could play in the three tissues. By bidirectional sequencing, we also found one nonsense mutation, 5 nonsynonymous, and 21 synonymous mutations in the coding region of the FMO3 gene in 11 duck breeds and some of them were predicted to be potentially associated with the activities of FMO3 protein.

Toll-like receptors (TLR) are trans-membrane sensors recognizing invading microbes. Toll-like receptors play a central role in initiating immune responses against several pathogens. In this study, we investigated the response of TLR and downstream genes to Marek's disease virus (MDV) infection. Forty 1-d-old chicks were randomly divided into 2 groups, with 20 chicks infected with MDV and 20 chicks mock-infected. Four chickens were euthanized respectively from infected and age-matched noninfected groups at 4, 7, 14, 21, and 28 d postinfection (dpi). Bursas, spleens, and thymuses were removed. The differential expression of TLR genes, including TLR3, TLR5, TLR7, TLR15, and TLR21, and downstream genes of TLR7, including MyD88, TRAF3, TRAF6, IFNA, IFNB, and IL6, in lymphoid tissues of MDV-infected and noninfected chickens was determined by real-time PCR. The results showed that the change of TLR genes was different in 3 lymphoid tissues. Expression of TLR7 and MyD88 was upregulated at 14 dpi and downregulated at 28 dpi in MDV-infected compared with noninfected spleens. The TRAF6 and IFNB were upregulated, and TRAF3, IFNA, and IL6 genes showed increasing trends in MDV-infected compared with noninfected spleens at 14 dpi. The expression of TLR3 and TLR15 genes was downregulated in MDV-infected compared with noninfected spleens at 28 dpi. The results indicated that TLR7 and its downstream genes were a response to MDV infection at 14 dpi. However, the function of TLR was impaired when the infection entered the tumor transformation phase. In bursas, TLR3 and TLR15 genes were upregulated at 7 and 4 dpi, respectively. It indicated that TLR3 and TLR15 might be involved in response to MDV infection in bursa at early phases. However, no differential expression of TLR genes was observed between MDV-infected and noninfected thymuses, which indicated that the thymus had little response to MDV infection mediated by TLR.

The MHM (male hypermethylated) region on the chicken Z chromosome is of interest in terms of sex determination both because hypermethylation of the promoter in males results in female-specific expression, and in females the non-coding transcripts accumulate in the region of a locus that encodes a key candidate gene for testis differentiation in birds - DMRT1. However, it has yet to be established whether MHM plays a role in either sex determination or gonadal development in chicken. Here, we sought to establish the MHM methylation pattern during chicken gonadal development and determine how this pattern correlates with the expression profiles of genes involved in sex determination and gonadal differentiation. As expected, we found that MHM methylation levels were higher in male than in female gonads at most stages of development. We also showed that there was no significant difference in the methylation patterns between the right and left gonad and that the pattern of methylation in female gonads was not affected by sex reversal. However, we did find an unexpected window where MHM promoter methylation levels were very low in both males and females. This developmental window coincided with a stage of gonad development where there is a dramatic increase in expression of key testis genes in males and of key ovary genes in females. The current study raises the possibility that the non-coding RNA transcripts of MHM may affect testis and ovary differentiation by influencing sex-specific gene expression.

Investigations on regulatory genes of feed intake will provide a rational scientific basis to improve future selection indices for more efficient chickens. In the present study, we investigated the association of 13 previously reported SNPs in the chicken adenosine monophosphate activated protein kinase (AMPK) subunits PRKAB1, PRKAG2, and PRKAG3 genes with body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in two distinct yellow meat-type strains. Six SNPs with a very low minor allele frequency were removed by genotype quality control and data filtering. The experimental population comprised 796 pedigreed males from two strains with different genetic backgrounds, 335 chickens from N202 and 461 chickens from N301. BW at 49 (BW49) and 70 days of age (BW70) and FI (from 49 to 70 days of age) were determined individually. BWG and FCR were computed based on BW and FI in the interval between 49 to 70 days. The results indicated that PRKAB1 SNPs rs14094358 and rs14094362 were significantly associated with BW70, BWG, and FI in the N202 strain, and rs14094361 and rs14094363 were significantly associated with FI and FCR in the N301 strain (P < 0.05). In addition, the PRKAG2 SNP rs14133282 showed significant association with FI in N202, and rs13535812 was significantly associated with BW70 in N202 (P < 0.05). Moreover, the PRKAG3 SNP rs13595570 was significantly associated with BW in N202 (P < 0.05), and significantly associated with FI and FCR in N301 (P < 0.05). Additionally, a two-SNP haplotype comprising rs14094361 and rs14094362 in PRKAB1 was significantly associated with BWG in N202 (P < 0.05). Meanwhile, haplotypes based on two SNPs, rs14133282, and rs13535812, showed significant effects on FI in N202 (P < 0.05). Our findings therefore provide important evidence for association of AMPK subunits polymorphisms with body weight, feed intake, and feed efficiency that may be applied in meat-type chicken breeding programs.

Suggestive SNPs associated with the earlobe color phenotype in Rhode Island Red chickens. (DOC 240 kb)

Chicken is considered to be an excellent model for genetic studies of phenotypic and genomic evolution, with large effective population size, specialized commercial lines, and strong human-driven selection. High-density chicken SNP chips can help to achieve a better understanding of the selection mechanisms in artificially selected populations. We performed the genome-wide tests for the selection signature in 385 White Leghorn hens and mapped positively selected regions to the genome annotations. Ten QTL related to egg production, egg quality, growth, and disease resistance traits were selected for extended haplotype homozygosity tests to give a brief overview of recent selection signatures in chicken QTL. We also reported 185 candidate genes/CDSs showing top P-values and slower decay of haplotype homozygosities. Some of these genes seemed to have significant effects on important economical traits, and most of them have not been reported in chickens. The current study provides a genome-wide map of linkage disequilibrium extents and distributions and selection footprints in the chicken genome. A panel of genes, including PRL, NCKX1, NRF1, LHX2, and SFRP1 associated with egg production, metabolism traits, and response to illumination were identified. In addition, there were more genes identified that have not yet been reported in chickens, and our results provide new clues for further study.

Epigenetic modification is widely known to be involved in embryo development, aging, tumorigenesis, and many complex diseases. Both hypermethylation of CpG islands at the gene promoters and global hypomethylation are involved in the initiation and progression of carcinogenesis. However, only a small portion of hypomethylation occurs at gene promoters and leads to the overexpression of certain oncogenes. To determine whether DNA methylation plays a role in tumorigenesis of Marek's disease, we selected one putative oncogene and 8 tumor suppressor genes from the gene expression profile for the analysis of DNA methylation variation. Four normal spleen tissues and 4 Marek's disease virus-infected tumor spleen tissues were collected, and the methylation level of the promoter region of each gene was analyzed using MassARRAY. As a result, the promoter region of CD30 was hypomethylated and displayed a significantly higher expression in Marek's disease virus-infected tumor spleen tissues compared with normal ones (P0.05). In neoplastic cells, CD30 was known to promote the survival and proliferation of T-cell lymphomas. This result suggests that activation of CD30 is possibly associated with the tumorigenesis of Marek's disease.

Marek's disease (MD) is an infectious disease of chickens caused by MD virus (MDV), which is a herpesvirus that initiates tumor formation. Studies have indicated that microRNAs (miRNAs) are linked with the development of cancers or tumors. Previously, gga-miR-130a was discovered downregulated in MDV-infected tissues. Here, we aimed to explore the further function of gga-miR-130a in MD. The expression of gga-miR-130a in MDV-infected and uninfected spleens was detected by quantitative real-time PCR (qRT-PCR). Subsequently, proliferation and migration assays of MDV-transformed lymphoid cells (MSB1) were carried out by transfecting gga-miR-130a. The target genes of gga-miR-130a were predicted using TargetScan and miRDB and clustered through Gene Ontology analysis. The target genes were validated by western blot, qRT-PCR, and a dual luciferase reporter assay. Our results show that the expression of gga-miR-130a was reduced in MDV-infected spleens. Gga-miR-130a showed an inhibitory effect on MSB1 cell proliferation and migration. Two target genes, homeobox A3 (HOXA3) and MyoD family inhibitor domain containing (MDFIC), were predicted and clustered to cell proliferation. Results indicate that gga-miR-130a regulates HOXA3 and MDFIC at the protein level but not at the mRNA level. Moreover, the gga-miR-130a binding sites of two target genes have been confirmed. We conclude that gga-miR-130a can arrest MSB1 cell proliferation and migration, and target HOXA3 and MDFIC, which are both involved in the regulation of cell proliferation. Collectively, gga-miR-130a plays a critical role in the tumorigenesis associated with chicken MD.

Background As a major economic trait in chickens, egg weight (EW) receives widespread interests in breeding, production and consumption. However, limited information is available for underlying genetic architecture of longitudinal trend in EW. Herein, we measured EWs at nine time points from onset of laying to 60 week of age, and conducted comprehensive genome-wide association studies (GWAS) in 1,534 F2 hens derived from reciprocal crosses between White Leghorn and Dongxiang chickens. Results Egg weights at all ages except the first egg weight (FEW) exhibited high SNP-based heritability estimates (0.47 ~ 0.60). Strong pair-wise genetic correlations (0.77 ~ 1.00) were found among all EWs. Nine separate univariate genome-wide screens suggested 73 signals showing significant associations with longitudinal EWs. After multivariate and conditional analyses, four variants on three chromosomes remained independent contributions. The minor alleles at two loci exerted consistent and positive substitution effects on EWs, and other two were negative. The four loci together accounted for 3.84 % of the phenotypic variance for FEW and 7.29 ~ 11.06 % for EWs from 32 to 60 week of age. We obtained five candidate genes, of which NCAPG harbors a non-synonymous SNP (rs14491030) causing a valine-to-alanine amino-acid substitution. Genome partitioning analysis indicated a strong linear correlation between the variance explained by each chromosome and its length, which provided evidence that EW follows a highly polygenic nature of inheritance. Conclusions Identification of significant genetic causes that together implicate EWs at different ages will greatly advance our understanding of the genetic basis behind longitudinal EWs, and would be helpful to illuminate the future breeding direction on how to select desired egg size. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1945-y) contains supplementary material, which is available to authorized users.

Summary To elucidate the origin and genetic structure of the domesticated duck in Eurasia and North America, we sequenced 114 duck D-loop sequences and retrieved 489 D-loop sequences from GenBank. In total, 603 ducks including 50 duck breeds/populations from eight countries (China, France, Russia, India, Kazakhstan, Mongolia, Thailand and USA) were used in this study. One hundred and thirty-four haplotypes and 81 variable sites were detected. H49 was the predominant haplotype, which was considered to be the same dominant haplotype found in the previous studies, and was found in 309 birds. The smallest values for both genetic differentiation index (FST, 0.04156) and the number of the net nucleotide substitutions between two populations (DA, 0.00018) were observed between Eurasian domestic ducks and Eurasian mallards. No geography, breed or population clusters were observed in the Eurasian domestic ducks and mallards. Five haplotypes were shared by USA mallards and Eurasian domestic duck/Eurasian mallards. Only one haplotype (H49) was shared by Eurasian domestic ducks and China spot-billed ducks. By combining phylogenetic analyses, haplotype network profile, genetic distances and shared haplotypes, we can draw two major conclusions: (i) Eurasian and North American mallards show a clear geographic distribution pattern; (ii) Eurasian domestic ducks are derived from the Eurasian mallards, not from the spot-billed ducks.

The objective of the current study was to analyze the methylation status of the chicken male hypermethylation (cMHM) region and mRNA expression levels of sex-dependent genes in adult female-to-male sex-reversed chickens. Sex reversal from genetic females into phenotypic males was induced by injection of 1.0 mg fadrozole, an aromatase inhibitor, into fertilized eggs at 3.0 days of incubation. Birds aged 30 weeks were classified into 4 groups according to the histological structure of their gonads and the natural logarithm of the ratio of testosterone to estradiol in serum, namely standard females, slightly sex-reversed females with left ovotestes, highly sex-reversed females with left testes, and standard males. The results showed that methylation of the cMHM amplicon was lowest in the ovaries of standard females and highest in testes of standard males. Methylation levels were significantly higher in the left testes of highly sex-reversed females than in the left ovotestes of slightly sex-reversed females (p < 0.05). Expression analysis of 9 sex-specific genes in the gonad indicated that DMRT1 and HINTZ had a similar expression pattern, with significantly higher levels in standard males as compared to standard females, slightly and highly sex-reversed females (p < 0.05). Expression of FOXL2, AMH, P450arom, SF1, and ERα was obviously higher in standard females than in standard males, slightly and highly sex-reversed females (p < 0.05). Expression of SOX9 in standard females was similar to that in slightly sex-reversed females and lower than in highly sex-reversed females and standard males (p < 0.05).

The objective of the present study was to evaluate the role of myofiber characteristics and the thickness of 2 major muscle membranes, perimysium and endomysium, in determining the breast meat tenderness of chickens. Birds from 2 breeds (White Leghorn and a line of broiler) were chosen. Chicks were sexed and wing-banded at hatch and were grown in separate cages in a single house. Sixty broilers and 60 White Leghorns were harvested at 6 wk of age, respectively, whereas another 60 White Leghorns were slaughtered at 18 wk of age. An equal number of males and females was maintained for each group. Body weight, breast muscle weight, pH, drip loss, cooking loss, Warner-Bratzler shear force value (SFV), total energy of shear force, fiber diameter, sarcomere length, myofiber density, and the thickness of endomysium and perimysium of the breast were determined for each bird. At 6 wk of age, histological examination indicated that the size of myofiber and thickness of endomysium and perimysium of broilers were larger than that those of White Leghorns (P < 0.01), whereas the SFV, drip loss, and cooking loss of broilers were smaller (P < 0.01). A comparison between the White Leghorns at 18 wk and the broilers at 6 wk, which were at similar BW but different ages, showed that the breast muscle weight of broilers was larger (P < 0.01) than that of White Leghorns. For breast muscle, the endomysium of broilers at 6 wk was thicker than that of White Leghorns at 18 wk (P < 0.01), whereas the perimysium was thinner (P < 0.01). The SFV, drip loss, and the cooking loss of broilers were smaller than those of White Leghorns at similar BW (P < 0.01). Meat tenderness was negatively correlated with myofiber density (-0.27) and the thickness of endomysium (-0.29) and positively correlated with the thickness of perimysium (0.20). It is suggested that muscle membrane should be considered in evaluating meat tenderness of the chicken.

The male hypemethylated (MHM) region, associated with sex-specific DNA methylation and histone acetylation, encodes non-coding RNAs that are expressed exclusively on Z chromosome in female chickens. The function of the MHM non-coding RNAs is unclear yet. In order to analyze the regulation effect of MHM on mRNA expression of sex-dependent genes, exogenous plasmid containing chicken MHM (cMHM) was constructed and injected into left testes of 13-week-old roosters. Comb color of the roosters in the pEGFP-N1-cMHM group became much paler, an indication of change in sex hormones, as compared with that of the negative control groups at the 10 days after injection. Real-time PCR was used to determine mRNA expression of sex-dependent genes in testes tissue. Transcription of ER alpha gene was significantly up-regulated in the pEGFP-N1-cMHM group compared with that of the pEGFP-N1 and normal saline groups (P0.01), while expression of DMRT1 mRNA declined severely in the pEGFP-N1-cMHM group compared with that of normal saline groups (P0.05). Nevertheless, expression of other candidate genes, such as AMH, FOXL2, SOX9, and P450arom did not change after injection of exogenous cMHM. In conclusion, down-regulation of DMRT1 and up-regulation of ER alpha in rooster testes treated with exogenous pEGFP-N1-cMHM plasmid might indicate a novel cMHM non-coding RNA-mediated mechanism in avian sex differentiation.

Blue-shelled eggs are gaining popularity as the consumption demand diversifies in some countries. This study was carried out to investigate the laying performance and egg quality of the blue-shelled egg layers as well as the effects of different housing systems on egg production and quality traits. One thousand pullets from Dongxiang blue-shelled layers were divided into 2 even groups and kept in different housing systems (outdoor vs. cage). Daily laying performance was recorded from 20 to 60 wk of age. External and internal egg quality traits were examined at 26, 34, 42, and 50 wk. Yolk cholesterol concentration and whole egg cholesterol content were measured at 40 wk of age. Average laying rate from 20 to 60 wk for the cage (54.7%) was significantly higher than that of outdoor layers (39.3%). Among all of the egg quality traits, only eggshell color was affected by housing system. Interaction between housing system and layer age was found in egg weight, eggshell color, eggshell ratio, yolk color, and yolk weight. Meanwhile, cholesterol concentration in yolk was 8.64 +/- 0.40 mg/g in the outdoor eggs, which was significantly lower than that of eggs from the cage birds (10.32 +/- 0.48 mg/g; P < 0.05). Whole egg cholesterol content in the outdoor eggs (125.23 +/- 6.32 mg/egg) was also significantly lower than that of eggs from the caged layers (158.01 +/- 8.62 mg/egg). The results demonstrated that blue-shelled layers have lower productivity in the outdoor system than in the cage system. Blue-shelled layers have lower egg weight, larger yolk proportion, and lower cholesterol content compared with commercial layers. In a proper marketing system, lower productivity could be balanced by a higher price for the better quality of blue-shelled eggs.

Polydactyly is one of the most common hereditary congenital limb malformations in chickens and other vertebrates. The zone of polarizing activity regulatory sequence (ZRS) is critical for the development of polydactyly. The causative mutation of polydactyly in the Silkie chicken has been mapped to the ZRS; however, the causative mutations of other chicken breeds are yet to be established. To understand whether the same mutation decides the polydactyly phenotype in other chicken breeds, we detected the single-nucleotide polymorphism in 26 different chicken breeds, specifically, 24 Chinese indigenous breeds and 2 European breeds. The mutation was found to have fully penetrated chickens with polydactyly in China, indicating that it is causative for polydactyly in Chinese indigenous chickens. In comparison, the mutation showed no association with polydactyly in Houdan chickens, which originate from France, Europe. Based on the different morphology of polydactyly in Chinese and European breeds, we assumed that the trait might be attributable to different genetic foundations. Therefore, we subsequently performed genome-wide association analysis (GWAS) to locate the region associated with polydactyly. As a result, a ~0.39 Mb genomic region on GGA2p was identified. The region contains six candidate genes, with the causative mutation found in Chinese indigenous breeds also being located in this region. Our results demonstrate that polydactyly in chickens from China and Europe is caused by two independent mutation events that are closely located in the chicken genome.

Genomic imprinting is a phenomenon characterized by parent-of-origin-specific gene expression. While widely documented in viviparous mammals and plants, imprinting in oviparous birds remains controversial. Because genomic imprinting is temporal- and tissue-specific, we investigated this phenomenon only in the brain tissues of 1-day-old chickens (Gallus gallus). We used next-generation sequencing technology to compare four transcriptomes pooled from 11 chickens, generated from reciprocally crossed families, to the DNA sequences of their parents. Candidate imprinted genes were then selected from these sequence alignments and subjected to verification experiments that excluded all but one SNP. Subsequent experiments performed with two new sets of reciprocally crossed families resulted in the exclusion of that candidate SNP as well. Attempts to find evidence of genomic imprinting from long non-coding RNAs yielded negative results. We therefore conclude that genomic imprinting is absent in the brains of 1-day-old chickens. However, due to the temporal and tissue specificity of imprinting, our results cannot be extended to all growth stages and tissue types.

Because it serves as the cytoplasm of the oocyte and provides a large amount of reserves, the egg yolk has biological significance for developing embryos. The ovary and its hierarchy of follicles are the main reproductive organs responsible for yolk deposition in chickens. However, the genetic architecture underlying the yolk and ovarian follicle weights remains elusive. Here, we measured the yolk weight (YW) at 11 age points from onset of egg laying to 72 weeks of age and measured the follicle weight (FW) and ovary weight (OW) at 73 weeks as part of a comprehensive genome-wide association study (GWAS) in 1,534 F2 hens derived from reciprocal crosses between White Leghorn (WL) and Dongxiang chickens (DX). For all ages, YWs exhibited moderate single nucleotide polymorphism (SNP)-based heritability estimates (0.25–0.38), while the estimates for FW (0.16) and OW (0.20) were relatively low. Independent univariate genome-wide screens for each trait identified 12, 3, and 31 novel significant associations with YW, FW, and OW, respectively. A list of candidate genes such as ZAR1, STARD13, ACER1b, ACSBG2, and DHRS12 were identified for having a plausible function in yolk and follicle development. These genes are important to the initiation of embryogenesis, lipid transport, lipoprotein synthesis, lipid droplet promotion, and steroid hormone metabolism, respectively. Our study provides for the first time a genome-wide association (GWA) analysis for follicle and ovary weight. Identification of the promising loci as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic yolk weight and ovarian follicle development and has practical significance in breeding programs for the alteration of yolk weight at different age points.

Background Heat stress has resulted in great losses in poultry production. To address this issue, we systematically analyzed chicken hypothalamus transcriptome responses to thermal stress using a 44 k chicken Agilent microarray, Methods Hypothalamus samples were collected from a control group reared at 25°C, a heat-stress group treated at 34°C for 24 h, and a temperature-recovery group reared at 25°C for 24 h following a heat-stress treatment. We compared the expression profiles between each pair of the three groups using microarray data. Results A total of 1,967 probe sets were found to be differentially expressed in the three comparisons with P < 0.05 and a fold change (FC) higher than 1.5, and the genes were mainly involved in self-regulation and compensation required to maintain homeostasis. Consistent expression results were found for 11 selected genes by quantitative real-time PCR. Thirty-eight interesting differential expression genes were found from GO term annotation and those genes were related to meat quality, growth, and crucial enzymes. Using these genes for genetic network analysis, we obtained three genetic networks. Moreover, the transcripts of heat-shock protein, including Hsp 40 and Hsp 90, were significantly altered in response to thermal stress. Conclusions This study provides a broader understanding of molecular mechanisms underlying stress response in chickens and discovery of novel genes that are regulated in a specific thermal-stress manner. Electronic supplementary material The online version of this article (doi:10.1186/s40104-015-0003-6) contains supplementary material, which is available to authorized users.

Background Feed contributes to over 60 % of the total production costs in the poultry industry. Increasing feed costs prompt geneticists to include feed intake and efficiency as selection goals in breeding programs. In the present study, we used an F2 chicken population in a genome-wide association study (GWAS) to detect potential genetic variants and candidate genes associated with daily feed intake (FI) and feed efficiency, including residual feed intake (RFI) and feed conversion ratio (FCR). Methods A total of 1534 F2 hens from a White Leghorn and Dongxiang reciprocal cross were phenotyped for feed intake and efficiency between 37 and 40 weeks (FI1, RFI1, and FCR1) and between 57 and 60 weeks (FI2, RFI2, and FCR2), and genotyped using the chicken 600 K single nucleotide polymorphism (SNP) genotyping array. Univariate, bivariate, and conditional genome-wide association studies (GWAS) were performed with GEMMA, a genome-wide efficient mixed model association algorithm. The statistical significance threshold for association was inferred by the simpleM method. Results We identified eight genomic regions that each contained at least one genetic variant that showed a significant association with FI. Genomic regions on Gallus gallus (GGA) chromosome 4 coincided with known quantitative trait loci (QTL) that affect feed intake of layers. Of particular interest, eight SNPs on GGA1 in the region between 169.23 and 171.55 Mb were consistently associated with FI in both univariate and bivariate GWAS, which explained 3.72 and 2.57 % of the phenotypic variance of FI1 and FI2, respectively. The CAB39L gene can be considered as a promising candidate for FI1. For RFI, a haplotype block on GGA27 harbored a significant SNP associated with RFI2. The major allele of rs315135692 was favorable for a lower RFI, with a phenotypic difference of 3.35 g/day between opposite homozygous genotypes. Strong signals on GGA1 were detected in the bivariate GWAS for FCR. Conclusions The results demonstrated the polygenic nature of feed intake. GWAS identified novel variants and confirmed a QTL that was previously reported for feed intake in chickens. Genetic variants associated with feed efficiency may be used in genomic breeding programs to select more efficient layers. Electronic supplementary material The online version of this article (doi:10.1186/s12711-015-0161-1) contains supplementary material, which is available to authorized users.

Background 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 (ESW), eggshell thickness (EST) and eggshell strength (ESS) at 11 time points from onset of laying to 72 weeks of age and conducted comprehensive genome-wide association studies (GWAS) in 1534 F2 hens derived from reciprocal crosses between White Leghorn (WL) and Dongxiang chickens (DX). Results ESWs at all ages exhibited moderate SNP-based heritability estimates (0.30 ~ 0.46), while the estimates for EST (0.21 ~ 0.31) and ESS (0.20 ~ 0.27) were relatively low. Eleven independent univariate genome-wide screens for each trait totally identified 1059, 1026 and 1356 significant associations with ESW, EST and ESS, respectively. Most significant loci were in a region spanning from 57.3 to 71.4 Mb of chromosome 1 (GGA1), which together account for 8.4 ~ 16.5 % of the phenotypic variance for ESW from 32 to 72 weeks of age, 4.1 ~ 6.9 % and 2.95 ~ 16.1 % for EST and ESS from 40 to 72 weeks of age. According to linkage disequilibrium (LD) and conditional analysis, the significant SNPs in this region were in extremely strong linkage disequilibrium status. Ultimately, two missense SNPs in GGA1 and one in GGA4 were considered as promising loci on three independent genes including ITPR2, PIK3C2G, and NCAPG. The homozygotes of advantageously effective alleles on PIK3C2G and ITPR2 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. Conclusions 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 (doi:10.1186/s12864-015-1795-7) contains supplementary material, which is available to authorized users.

Manhattan plots (left) and quantile-quantile plots (right) for first egg weight (FEW) and egg weights at 32, 40, 44, 48, 52, 56 and 60 wk of age (EW32 and EW40~EW60). The Manhattan plots indicate -log10 (observed P-values) for genome-wide SNPs (y-axis) plotted against their respective positions on each chromosome (x-axis), and the horizontal green and black lines depict the genome-wide suggestive (1.69 × 10-5) and significant (8.43 × 10-7) threshold, respectively. For quantile-quantile plots, the x-axis shows the expected -log10-transformed P-values, and the y-axis represents the observed -log10-transformed P-values. The raw and adjusted genomic inflation factors (λ and λ_1000) are shown on the top left in the QQ plot. (A) ~ (H) indicate egg weights at eight different wk of age, respectively. (PDF 842 kb)

China is rich in chicken genetic resources, and many indigenous breeds can be found throughout the country. Due to poor productive ability, some of them are threatened by the commercial varieties from domestic and foreign breeding companies. In a large-scale investigation into the current status of Chinese poultry genetic resources, 78 indigenous chicken breeds were surveyed and their blood samples collected. The genomes of these chickens were screened using microsatellite analysis. A total of 2740 individuals were genotyped for 27 microsatellite markers on 13 chromosomes. The number of alleles of the 27 markers ranged from 6 to 51 per locus with a mean of 18.74. Heterozy-gosity ( H ) values of the 78 chicken breeds were all more than 0.5. The average H value (0.622) and polymorphism information content (PIC, 0.573) of these breeds suggested that the Chinese indige-nous chickens possessed more genetic diversity than that reported in many other countries. The fixa-tion coefficients of subpopulations within the total population ( F ST ) for the 27 loci varied from 0.065 (LEI0166) to 0.209 (MCW0078), with a mean of 0.106. For all detected microsatellite loci, only one (LEI0194) deviated from Hardy-Weinberg equilibrium (HWE) across all the populations. As genetic drift or non-random mating can occur in small populations, breeds kept on conservation farms such as Langshan chicken generally had lower H values, while those kept on large populations within con-servation regions possessed higher polymorphisms. The high genetic diversity in Chinese indigenous breeds is in agreement with great phenotypic variation of these breeds. Using Nei’s genetic distance and the Neighbor-Joining method, the indigenous Chinese chickens were classified into six categories that were generally consistent with their geographic distributions. The molecular information of genetic diversity will play an important role in conservation, supervision, and utilization of the chicken re-sources.

One of the main concerns for poultry producers is how to maintain egg uniformity and stability in size and weight following the rapid growth during the early laying period. In this study, we aimed to investigate the increase in egg weight with advancing hen age, and to estimate genetic parameters of these increment traits in 2 pure lines of chickens (i.e., 2,010 White Leghorn and 1,200 brown-egg dwarf hens), using the restricted maximum likelihood method with the DMU procedure. We collected age at first egg (AFE), first egg weight (FEW) and kept records of egg weight per 10 wk from 30 to 60 wk of age. Meanwhile, the increments of egg weight were calculated for the evaluation of age-dependent dynamic changes. The increment of egg weight gained dramatically before 30 wk of age and became slower with the advance of age. Heritability estimates of AFE were larger than 0.32, and the low to moderate genetic correlations between AFE and FEW were observed in the 2 lines. The FEW showed high variation level compared with egg weights at later ages in the 2 lines, and had moderate heritability estimates in White Leghorns (0.20) and dwarf hens (0.33). Egg weights at different ages were highly heritable in the 2 lines (h(2) ≥ 0.35), and had strong genetic and phenotypic correlations among different ages. The estimates of heritability for most increment traits were low to moderate, especially those increments for 10-wk intervals ranging from 0.00 to 0.14. The genetic correlations among 3 consecutive egg weight increments for 10-wk intervals were low to moderate. Our results in the 2 lines should provide important insights into the genetic architecture of increment traits and offer some suggestions for producing uniform and stable eggs in response to advancing age.

Insertion and deletion (INDEL) is one of the main events contributing to genetic and phenotypic diversity, which receives less attention than SNP and large structural variation. To gain a better knowledge of INDEL variation in chicken genome, we applied next generation sequencing on 12 diverse chicken breeds at an average effective depth of 8.6. Over 1.3 million non-redundant short INDELs (1-49 bp) were obtained, the vast majority (92.48%) of which were novel. Follow-up validation assays confirmed that most (88.00%) of the randomly selected INDELs represent true variations. The majority (95.76%) of INDELs were less than 10 bp. Both the detected number and affected bases were larger for deletions than insertions. In total, INDELs covered 3.8 Mbp, corresponding to 0.36% of the chicken genome. The average genomic INDEL density was estimated as 0.49 per kb. INDELs were ubiquitous and distributed in a non-uniform fashion across chromosomes, with lower INDEL density in micro-chromosomes than in others, and some functional regions like exons and UTRs were prone to less INDELs than introns and intergenic regions. Nearly 620,253 INDELs fell in genic regions, 1,765 (0.28%) of which located in exons, spanning 1,358 (7.56%) unique Ensembl genes. Many of them are associated with economically important traits and some are the homologues of human disease-related genes. We demonstrate that sequencing multiple individuals at a medium depth offers a promising way for reliable identification of INDELs. The coding INDELs are valuable candidates for further elucidation of the association between genotypes and phenotypes. The chicken INDELs revealed by our study can be useful for future studies, including development of INDEL markers, construction of high density linkage map, INDEL arrays design, and hopefully, molecular breeding programs in chicken.

MicroRNA (miRNA) are a class of highly conserved, small noncoding RNA that emerge as key posttranscriptional regulators in various neoplastic transformations. Our previous study profiling the miRNA transcriptome in Marek's disease virus (MDV)-induced lymphoma revealed many novel and differentially expressed miRNA, including gga-miR-26a, which was downregulated in MDV-infected spleens of chickens. In this study, differential expression of gga-miR-26a between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection was analyzed by real-time PCR. The results showed gga-miR-26a were downregulated in MDV-infected spleens at cytolytic infection, latency, and tumor transformation phases. Subsequent cell proliferation assay revealed cell viability was lower in gga-miR-26a mimic transfection group than that in negative controls. Target genes of gga-miR-26a were identified by luciferase reporter gene assay. The results showed significant interaction between gga-miR-26a and Never In Mitosis Gene A (NIMA)-related kinase 6 (NEK6) gene. Subsequent gain of function experiment and Western blot assay showed that mRNA and protein levels of NEK6 were downregulated after gga-miR-26 mimic was transfected into MDV-transformed lymphoid cell line (MSB-1), indicating that NEK6 was modulated by gga-miR-26a. The expression of NEK6 showed a higher trend in MDV-infected samples including tumorous spleen and MD lymphoma from liver than that in noninfected controls. The results suggested that gga-miR-26a inhibited MSB-1 cell proliferation. Gga-miR-26a and its direct target, NEK6, might play important roles in MDV infection.

Protoporphyrin IX (PpIX), an immediate precursor of heme, is the main pigment resulting in the brown coloration of eggshell. The brownness and uniformity of the eggshell are important marketing considerations. In this study, 9 chickens laying darker brown shelled eggs and 9 chickens laying lighter brown shelled eggs were selected from 464 individually caged layers in a Rhode Island Red pureline. The PpIX contents were measured with a Microplate Reader at the wavelength of 412 nm and were compared in different tissues of the 2 groups. Although no significant difference in serum, bile, and excreta was found between the 2 groups, PpIX content in the shell gland and eggshell of the darker group was higher than in those of the lighter group, suggesting that PpIX was synthesized in the shell gland. We further determined the expression levels of 8 genes encoding enzymes involved in the heme synthesis and transport in the liver and shell gland at 6 h postoviposition by quantitative PCR. The results showed that expression of aminolevulinic acid synthase-1 (ALAS1) was higher in the liver of hens laying darker brown shelled eggs, whereas in the shell gland the expression levels of ALAS1, coproporphyrinogen oxidase (CPOX), ATP-binding cassette family members ABCB7 and ABCG2, and receptor for feline leukemia virus, subgroup C (FLVCR) were significantly higher in the hens laying darker brown shelled eggs. Our results demonstrated that hens laying darker brown shelled eggs could deposit more PpIX onto the eggshell and the brownness of the eggshell was dependent on the total quantity of PpIX in the eggshell. More heme was synthesized in the liver and shell gland of hens laying darker brown shelled eggs than those of hens laying lighter brown shelled eggs. High expression level of ABCG2 might facilitate the accumulation of PpIX in the shell gland.

The chicken major histocompatibility complex (MHC) has abundant SNP and indels, and is closely related with host genetic resistance or susceptibility to disease. The LEI0258 locus is the most variable in the MHC region, and is a useful marker in reflecting the variability of MHC. In this study, we applied the LEI0258 microsatellite marker to investigate polymorphism of MHC in Chinese indigenous chickens. The size of LEI0258 fragments in 1,617 individuals from 33 Chinese chicken breeds was detected by capillary electrophoresis, and 213 samples with different fragment sizes were further sequenced. A total of 69 alleles ranging from 193 to 489 bp were found, including 21 novel alleles and 28 private alleles that existed in only one breed. Three alleles, 249 bp (7.04%), 489 bp (6.57%), and 309 bp (6.10%), were the most frequent in the indigenous chickens. A 489-bp novel allele was unique in Chinese local chicken breeds. Three indels and 4 SNP of upstream/downstream of 2 repeat regions (R13/R12) were found. Abundant variations indicate high genetic diversity at the MHC region in indigenous chickens. Rare alleles are vulnerable to genetic drift in small populations, and can be used as molecular markers for monitoring the dynamic conservation of many indigenous breeds.

DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively.

Muscle characteristics such as myofiber diameter, density, and total number are important traits in broiler breeding and production. In the present study, 19 SNP of 13 major genes, which are located in the vicinity of quantitative trait loci affecting breast muscle weight, including INS, IGF2, PIK3C2A, AKT3, PRKAB2, PRKAG3, VEGFA, RPS6KA2/3, FIGF, and TGF-β1/2/3, were chosen to be genotyped by high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in a broiler population. One hundred twenty birds were slaughtered at 6 wk of age. Body weight, breast muscle weight, myofiber diameter, density, and total number were determined for each bird. Six SNP with a very low minor allele frequency (1%) were excluded for further analysis. The remaining 13 SNP were used for the association study with muscle characteristics. The results showed that SNP in TGF-β1/2/3 had significant effects on myofiber diameter. A SNP in PRKAG3 had a significant effect on myofiber density (P0.05). A CG mutation in FIGF was strongly associated with total fiber number (P0.05). Additionally, birds with the GG genotype of the CG mutation in AKT3 had significantly larger myofiber numbers (P0.05) than birds with the CC or GC genotype. The SNP identified in the present study might be used as potential markers in broiler breeding.

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F2 chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level., Author Summary The eggshell color of birds is of wide interest, but the molecular basis remained unknown until our discovery, reported here. The blue eggshell is found not only in wild birds but also in domestic fowls. In this study, we identified that blue eggshell in chickens from different geographical regions is caused by a ∼4.2 kb EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion in chicken is a derived mutation in domestic chickens. The genetic determination of blue eggshell in other birds requires further investigation. We also found that the EAV-HP insertions in the chickens from China and America were separate integration events, which presents us with a parallel molecular evolution example driven by artificial selection.

Marek's disease is a viral neoplastic disease of chickens caused by Marek's disease virus (MDV). Gene expression patterns have been investigated at different MDV infection stages, but there is limited research about the late tumor transformation phase. In this experiment, 44K Agilent chicken genome-wide expression microarrays were used to profile differential expression in tumorous spleens (TS) from severely morbid chickens and apparently normal spleens from survivors (SS) after MDV infection and expression in noninfected spleens (NS) from controls. There were 4,317 differentially expressed (DE) genes in TS versus NS. However, no DE genes were detected in SS versus NS, suggesting that maintenance of, or return to, homeostasis of gene activity in survivor spleens. Downregulated genes in tumorous spleens mainly enriched in the cytokine-cytokine receptor interaction pathway, and commonly investigated genes in Marek's disease study, IL6, IL18, IFNA, and IFNG were nondifferentially expressed, which indicates host inflammatory response was impaired. The IL10 and TNFRSF8 genes were upregulated in tumorous spleens. We speculated that IL10 might be exploited by MDV to escape from host immune surveillance, as reported for Epstein-Barr virus, which stimulated T cells secreting IL10 to subvert immune response. Previous study reported that transcription from TNFRSF8 promoter could be enhanced by MDV oncogene Meq. In this study, the increased expression of TNFRSF8 indicated interaction between MDV and TNFRSF8, which might facilitate pathogenesis and tumor transformation. The expression of many members in IGF system was changed in tumorous compared with noninfected spleens. The downregulation of IGFBP7 was considered to be associated with MD lymphoma transformation. Gene expression change of multiple regulatory pathways indicated their involvements in facilitating tumor transformation.

The correlation between IgY levels of the serum and the yolk has been well documented in wild and domestic birds. The levels of total yolk IgY can be an index of the general health status of birds and may contribute to breeding programs when fitness of the offspring is a concern. We measured the levels of total serum IgY and yolk IgY in three different breeds (White Leghorn, Silkie and Dongxiang blue-shell) using indirect ELISA, and found that there was a significantly positive correlation between the levels of total serum IgY and total yolk IgY in all three breeds (White Leghorn: r = 0.404, P P P P P

Summary Marek's disease (MD) is a neoplastic disease in chickens, caused by the Marek's disease virus (MDV). To investigate host genetic resistance to MD, we conducted a genome-wide association study (GWAS) on 67 MDV-infected chickens based on a case and control design, including 57 susceptible chickens in the case group and 10 resistant chickens as controls. After searching 38 655 valid genomic markers, two SNPs were found to be associated with host resistance to MD. One SNP, rs14527240, reaching chromosome-wide significance level (P

Marek’s disease is a lymphoproliferative neoplastic disease of the chicken, which poses a serious threat to poultry health. Marek’s disease virus (MDV)-induced T-cell lymphoma is also an excellent biomedical model for neoplasia research. Recently, miRNAs have been demonstrated to play crucial roles in mediating neoplastic transformation. To investigate host miRNA expression profiles in the tumor transformation phase of MDV infection, we performed deep sequencing in two MDV-infected samples (tumorous spleen and MD lymphoma from liver), and two non-infected controls (non-infected spleen and lymphocytes). In total, 187 and 16 known miRNAs were identified in chicken and MDV, respectively, and 17 novel chicken miRNAs were further confirmed by qPCR. We identified 28 down-regulated miRNAs and 11 up-regulated miRNAs in MDV-infected samples by bioinformatic analysis. Of nine further tested by qPCR, seven were verified. The gga-miR-181a, gga-miR-26a, gga-miR-221, gga-miR-222, gga-miR-199*, and gga-miR-140* were down-regulated, and gga-miR-146c was up-regulated in MDV-infected tumorous spleens and MD lymphomas. In addition, 189 putative target genes for seven differentially expressed miRNAs were predicted. The luciferase reporter gene assay showed interactions of gga-miR-181a with MYBL1, gga-miR-181a with IGF2BP3, and gga-miR-26a with EIF3A. Differential expression of miRNAs and the predicted targets strongly suggest that they contribute to MDV-induced lymphomagenesis.

Some members of the low-density lipoprotein receptor (LDLR) family play important roles in the regulation of lipoprotein metabolism and egg quality traits. Low-density lipoprotein receptor-related protein 2 (LRP2) gene belongs to the LDLR super family, and widely expresses in many tissues. This work identified and genotyped 1 single-nucleotide polymorphism (SNP), T14347C, at 3'-UTR of the LRP2 using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and analyzed the effects of the SNP (T14347C) on egg-quality traits in 544 dwarf hens from 44 sire families. Frequencies of this SNP in the studied population did not agree with the Hardy-Weinberg equilibrium (P < 0.0001). Egg weight, albumen weight, albumen height, and albumen ratio of the TT genotype were significantly higher than those of the CC genotype (P < 0.05), whereas eggshell ratio of the TT genotype was significantly lower than that of the CC genotype (P < 0.05). The relative expression level of the LRP2 gene in the magnum was determined by real-time quantitative PCR. The gene expression of genotype CC individuals was significantly higher than that of TT and CT birds (P < 0.05). By combining both genetic effects and expression analyses results, we propose that the LRP2 gene is a good candidate gene, exhibiting a key role in albumen formation processes.

Major histocompatibility complex has previously been shown to influence the resistance of chicken to Marek's disease virus (MDV). However, little is known about expression of other genes in the MHC-I and II pathway after MDV infection. This study aimed at investigating 8 immune-related genes in the MHC core region that affects host responses to MDV. Spleens of infected and age-matched uninfected chickens were removed at 4, 7, 14, 21, and 28 d postinfection for gene expression detection using real-time PCR. Different expression patterns of MHC-I and II pathway genes were observed in the spleen. In the MHC-I pathway, the expression of transporter of antigen protein 1 (TAP1), transporter of antigen protein 2 (TAP2), and transporter of antigen protein-binding protein (TAPBP) genes was significantly increased in the spleen of MDV-infected than that of uninfected chickens. It indicated that host antivirus responses were generated to enhance antigen presentation. However, MHC-II pathway genes showed contrary trends. Classical MHC-II β chain major gene (BLB2) and nonclassical class II genes [DM α chain gene (DMA), DM β chain gene-1 (DMB1), and DM β chain gene-2 (DMB2)] had consistent lower transcripts in spleens of MDV-infected than that of uninfected chickens, which reflected that MDV interfered with multiple components of the MHC-II pathway. Overall, expression of most genes in the MHC core region was altered; moreover, the genes in endogenous and exogenous antigen presentation pathways had different expression patterns in the spleen after MDV infection.

Low-density lipoprotein receptor-related protein 8 (LRP8), a member of the low-density lipoprotein receptor gene family with a role in clusterin processing, was investigated as a candidate gene for egg quality-related traits. One SNP from C to T at position 1623 of the open reading frame of LRP8 was identified and genotyped by a high-throughput genotyping method, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in 747 egg-type dwarf layers from 44 sire families. There were no significant differences among genotypes for any interior egg traits measured, except for yolk color, in which color was deeper for the TT genotype than CC or CT (P0.05). For shell traits, strength and thickness were greater for TT than CC (P0.05), with CT intermediate and not different from either. Shape index was lower for CT than either TT or CC, which did not differ, whereas for shell color, CT was intermediate to the homozygotes, which differed (CCTT). The present results indicated that LRP8, as a new member of eggshell matrix protein, may be a candidate gene associated with eggshell traits.

Injection of an aromatase inhibitor before gonadal sex differentiation can induce female-to-male sex reversal in chicken, but a higher dose could reduce hatchability. In the present study, the dosage effect of Fadrozole, a nonsteroidal aromatase inhibitor, on gonadal differentiation was analyzed in sex-reversed genetic female chicken. There was no significant difference for the hatchability among low-dose (0.1, 0.3 and 0.5 mg) Fadrozole-injected groups and control groups (p > 0.05), but in high-dose groups (1.0 mg and 1.3 mg) the hatchability was significantly reduced (p < 0.05). Complete phenotypic sex reversal was observed in both high- and low- dose groups, with more than 90% of genetic females reversed into phenotypic male chicks. The degree of gonadal differentiation in 124 chicken at 30 weeks of age was classified into 5 categories according to the visual appearance and histological structure of the gonads. Category I and V were standard ovary and testis, respectively, while category II, III, and IV represented gonads of slightly, moderately, and highly sex-reversed females, respectively. Most gonads in low-dose groups were classified into category I, II, and III, while those in high-dose groups were mainly category IV. The serum testosterone levels in the 1.3 mg group were significantly higher and the estradiol levels lower than those in low-dose groups (p < 0.05). In conclusion, the range from 0.5 to 1.0 mg Fadrozole is a key interval for survival of chicken embryos. The gonadal differentiation in sex-reversed females at 30 weeks of age is highly dependent on the dose of Fadrozole injected prior to gonadal sex differentiation.

Mx proteins are interferon-induced GTPases that confer antiviral activities against RNA viruses. We analysed the molecular evolution of the Mx gene in birds using data on interspecific divergence in anseriform and galliform birds, and on intraspecific diversity in commercial chicken lines, local Chinese chicken breeds as well as in the mallard. The overall ratio of non-synonymous to synonymous substitution was unusually high, 0.80, indicating relaxed constraint or positive selection. Evidence for the latter was provided by that a total of 11-18 codons were found to have evolved under positive selection. The great majority of these codons are located in a region unique to birds at the N-terminal end of the Mx protein. We found an excess of non-synonymous polymorphisms relative to synonymous variants in all comparisons. This, together with positive Tajima's D values in the local Chinese chicken breeds and in the mallard suggests that balancing selection is acting in avian Mx genes. As such, Mx mimics the major histocompatibility complex system, indicating that heterozygous individuals are better off withstanding pathogen attack.

Gamete-recognition proteins have been shown to evolve by positive selection in diverse organism groups, such as marine invertebrates and mammals, although underlying evolutionary mechanisms driving this rapid divergence are poorly understood. However, several hypotheses have been put forward to explain the observed pattern, including different forms of sexual conflict and sperm competition. Because female gametes require more energy to produce than male gametes, female organisms suffer more when fertilisation goes wrong. One process that results in a failed mammalian fertilisation is polyspermy, when >1 sperm fertilises the egg. However in birds, there is no such sexual conflict because multiple sperm typically bind and fuse with the egg. If sexual conflict driven by polyspermy avoidance is important for the evolution of gamete-recognition proteins in vertebrates, we expect to find positive selection in the genes to be less pronounced in birds. We therefore sequenced six genes (ZP1, ZP2, ZP4, ZPAX, CD9, and Acrosin) encoding gamete-recognition proteins in several bird species to test for positive selection. For comparison, we also analysed ortologous sequences in a set of mammalian species. We found no major differences in the occurrence of adaptive evolution and the strength of selection between bird and mammal orthologs. From this we conclude that polyspermy avoidance does not act as the main underlying evolutionary force shaping the rate of evolution in these genes. We discuss other possible processes that could explain positive selection of gamete-recognition proteins in birds and mammals, such as hybridisation avoidance, cryptic female choice, and postcopulatory sperm competition.

In order to elucidate the domestication history of Peking ducks, 190 blood samples from six Chinese indigenous duck breeds were collected with 186 individuals genotyped by 15 microsatellite markers. Both the F(ST) and Nei's standard genetic distances (D(s)) from the microsatellite data indicated high genetic differentiation between Peking duck and other Chinese indigenous breeds. The haplotype network with mtDNA data showed that most of the Peking duck haplotypes were distinctly different from those of other domestic breeds. Although the H01 haplotype was shared by all domesticated duck breeds, Peking ducks displayed 12 specific domestic duck haplotypes, including four similar haplotypes H02, H04, H08 and H22, that formed a single haplogroup (A). Both H02 and H22 haplotypes were also shared by mallard and Peking ducks, indicating that Peking ducks originated from wild mallard ducks.

The Mx protein, which confers resistance to orthomyxovirus, has been detected in several organisms, and one nonsynonymous substitution (S631N) of the chicken Mx protein has been shown to affect resistant activities to the avian influenza virus in vitro. In the current study, the genomic sequence and polymorphism of the chicken Mx gene are reported. The full length of the chicken Mx gene spans about 21 kb, with 13 exons on chromosome 1 of the chicken genome. A total of 237 single nucleotide polymorphisms were found in the chicken Mx gene by comparison among 4 directly sequenced Mx genomic DNA sequences, and the reference sequence was inferred from the chicken genome project. The genomic diversity of the chicken Mx gene showed large variation in different regions, with the highest diversity in the 5' untranslated region and the lowest in the 3' untranslated region. The genomic structure and variation of sequences gathered here will allow an extensive analysis of the gene function with the aim of improving the antiviral resistance activities of chickens.

Since the feed cost is a major determinant of profitability in poultry industry, how to improve feed efficiency through genetic selection is an intriguing subject for breeders and producers. As a more suitable indicator assessing feed efficiency, residual feed intake (RFI) is defined as the difference between observed and expected feed intake based on maintenance and growth. However, the genetic mechanisms responsible for RFI in chickens are still less well appreciated. In this study, we investigated the duodenal transcriptome architecture of extreme RFI phenotypes in the six brown-egg dwarf hens (three per group) using RNA sequencing technology. Among all mapped reads, an average of 75.62% fell into annotated exons, 5.50% were located in introns, and the remaining 18.88% were assigned to intergenic regions. In total, we identified 41 promising candidate genes by differential expression analysis between the low and high RFI groups. Furthermore, qRT-PCR assays were designed for 10 randomly chosen genes, and nine (90.00%) were successfully validated. Functional annotation analyses revealed that these significant genes belong to several specific biological functions related to digestibility, metabolism and biosynthesis processes as well as energy homeostasis. We also predicted 253 intergenic coding transcripts, and these transcripts were mainly involved in fundamental biological regulation and metabolism processes. Our findings provided a pioneering exploration of biological basis underlying divergent RFI using RNA-Seq, which pinpoints promising candidate genes of functional relevance, is helpful to guide future breeding strategies to optimize feed efficiency and assists in improving the current gene annotation in chickens.

The Mx gene is considered to confer positive antiviral responses to the orthomyxovirus in many organisms. In the chicken, 1 nonsynonymous single nucleotide polymorphism (G to A) at position 2,032 of Mx cDNA was demonstrated to confer positive antiviral activity in vitro to avian influenza virus in a previous study. In the current study, 15 Chinese native chicken breeds, 4 highly selected commercial lines, and the Red Jungle Fowl were selected to detect allele frequencies of the Mx mutation. The frequencies of the favorable allele A in native breeds were 0.7241 to 0.9554, which were much higher than those (0.0565 to 0.2742) found in the commercial populations. Whereas most native breeds were in Hardy-Weinberg equilibrium at this locus (P > 0.01), 3 out of 4 commercial populations were not in Hardy-Weinberg equilibrium (P < 0.01). Selection, environment, and negative correlations between production and disease resistant traits could contribute to highly skewed frequencies of the mutation among native breeds and commercial populations. The results suggested that further studies are needed with regard to the genetic resistance to avian influenza in different populations with various domestication background and selection history.