Eggshell translucency in late-phase laying hens and its effect on egg quality and physiological indicators.

Eggshell translucency severely affects external egg quality, and variations in the eggshell or eggshell membrane are considered the structural basis of the trait. Research has shown that 1.85% additional mixed fatty acids in the diet would greatly decrease the occurrence of eggshell translucency. Only a few studies have examined the phenotypic regularity of eggshell translucency with the increasing age of hens. Therefore, two strains, 1139 Rhode Island Red-White (RIR-White) and 836 Dwarf Layer-White (DWL-White), were used, and from each strain, 30 hens each that consecutively laid translucent or opaque eggs at 67 wks of age were selected. Subsequently, eggshell translucency, internal quality and external quality of eggs, and total cholesterol, albumin, calcium binding protein and other physiological indicators related to lipid, lipoprotein, and calcium metabolisms at the 75th, 79th, and 83rd wks of age in the late phase of the laying cycle were determined. Results: (1) In terms of flocks, for both strains, the translucency scores of the translucent groups were significantly higher than those of the opaque groups (P < 0.05); in terms of individuals, 81.1% RIR-White and 82.8% DWL-White hens consecutively laid eggs of the same or similar translucency, indicating the stability of the trait with increasing hen age; (2) In RIR-White, the eggshell strength of the translucent group at 75 weeks was significantly higher than that of the opaque group (P < 0.05); in DWL-White, the eggshell membrane thickness of the translucent group at the 75th and 83rd weeks was significantly lower than that of the opaque group (P < 0.05); (3) Compared to the opaque groups, the translucent groups had lower total cholesterol content in both RIR-White and DWL-White, lower albumin content in DWL-White at the 79th weeks (P < 0.05), and higher calcium-binding protein (CALB1) in RIR-White at the 83rd weeks (P < 0.05). In summary, this study illustrates the stability of eggshell translucency in late-phase laying hens and provides a reference of physiological indicators for exploring the formation of translucent eggs.

Comprehensive analysis of structural variants in chickens using PacBio sequencing.

Structural variants (SVs) are one of the main sources of genetic variants and have a greater impact on phenotype evolution, disease susceptibility, and environmental adaptations than single nucleotide polymorphisms (SNPs). However, SVs remain challenging to accurately type, with several detection methods showing different limitations. Here, we explored SVs from 10 different chickens using PacBio technology and detected 49,501 high-confidence SVs. The results showed that the PacBio long-read detected more SVs than Illumina short-read technology genomes owing to some SV sites on chromosomes, which are related to chicken growth and development. During chicken domestication, some SVs beneficial to the breed or without any effect on the genomic function of the breed were retained, whereas deleterious SVs were generally eliminated. This study could facilitate the analysis of the genetic characteristics of different chickens and provide a better understanding of their phenotypic characteristics at the SV level, based on the long-read sequencing method. This study enriches our knowledge of SVs in chickens and improves our understanding of chicken genomic diversity.

A ∼ 4.1 kb deletion in IRX1 gene upstream is completely associated with rumplessness in Piao chicken.

Piao chicken, a Chinese indigenous rumpless chicken breed, lacks pygostyle, caudal vertebra, uropygial gland and tail feathers. The rumplessness in Piao chicken presents an autosomal dominant inheritance pattern. However, the molecular genetic mechanisms underlying the rumplessness in Piao chicken remains unclear. In this study, whole-genome resequencing was performed for 146 individuals from 10 chicken breeds, including 9 tailed chicken breeds and Piao rumpless breed. Tailbone CT scan for Piao chickens and WL chickens, revealed that some Piao chicken tails were normal in number, and for a few Piao chickens tail length and tail bone numbers were between the rumpless and the normal tailed chickens. The results showed that the rumpless phenotype has not been completely fixed in Piao chicken breed. Using selection signature analysis and structural variation detection, we found a 4174 bp deletion located in the upstream region of IRX1 gene on chromosome 2 related to rumpless phenotype. Structural variation genotyping showed that the deletion was present in all 32 rumpless Piao chickens (del/del, wild/del) and absent from all 112 tailed chickens included in the dataset for the other 9 breeds and 2 tailed Piao chickens (wild/wild). In summary, all rumpless Piao chickens tested here carry this deletion mutation, to show a complete linkage association with rumplessness trait. We suggested that the 4174 bp deletion could be causative for rumpless phenotype in Piao chicken since this is the only mutation to show the complete linkage disequilibrium with rumplessness on whole genome level across all of 146 chickens from the 10 breeds. This study could facilitate a better understanding of the genetic characteristics of Piao chicken.

Genomic and transcriptomic analyses reveal genetic adaptation to cold conditions in the chickens.

Under the pressure of natural and artificial selection, domestic animals, including chickens, have evolved unique mechanisms of genetic adaptations such as high-altitude adaptation, hot and arid climate adaptation, and desert adaptation. Here, we investigated the genetic basis of cold tolerance in chicken by integrating whole-genome and transcriptome sequencing technologies. Genome-wide comparative analyses of 118 chickens living in different latitudes showed 46 genes and several pathways that may be involved in cold adaptation. The results of the functional enrichment analysis of differentially expressed genes proved the important role of metabolic pathways and immune-related pathways in cold tolerance in chickens. The subsequent integration of whole genome and transcriptome sequencing technology further identified six genes - dnah5 (dynein axonemal heavy chain 5), ptgs2 (prostaglandin-endoperoxide synthase 2), inhba (inhibin beta A subunit), irx2 (iroquois homeobox 2), ensgalg00000054917, and ensgalg00000046652 - requiring more detailed studies. In addition, we also discovered different allele frequency distributions of five SNPs (single nucleotide polymorphisms) within ptgs2 and nine SNPs within dnah5 in chickens in different latitudes, suggesting strong selective pressure of these two genes in chickens. We provide a novel insight into the genetic adaptation in chickens to cold environments, and provide a reference for evaluating and developing adaptive chicken breeds in cold environments.

Identification of candidate genomic regions for chicken egg number traits based on genome-wide association study.

BACKGROUND: Since the domestication of chicken, various breeds have been developed for food production, entertainment, and so on. Compared to indigenous chicken breeds which generally do not show elite production performance, commercial breeds or lines are selected intensely for meat or egg production. In the present study, in order to understand the molecular mechanisms underlying the dramatic differences of egg number between commercial egg-type chickens and indigenous chickens, we performed a genome-wide association study (GWAS) in a mixed linear model. RESULTS: We obtained 148 single nucleotide polymorphisms (SNPs) associated with egg number traits (57 significantly, 91 suggestively). Among them, 4 SNPs overlapped with previously reported quantitative trait loci (QTL), including 2 for egg production and 2 for reproductive traits. Furthermore, we identified 32 candidate genes based on the function of the screened genes. These genes were found to be mainly involved in regulating hormones, playing a role in the formation, growth, and development of follicles, and in the development of the reproductive system. Some genes such as NELL2 (neural EGFL like 2), KITLG (KIT ligand), GHRHR (Growth hormone releasing hormone receptor), NCOA1 (Nuclear receptor coactivator 1), ITPR1 (inositol 1, 4, 5-trisphosphate receptor type 1), GAMT (guanidinoacetate N-methyltransferase), and CAMK4 (calcium/calmodulin-dependent protein kinase IV) deserve our attention and further study since they have been reported to be closely related to egg production, egg number and reproductive traits. In addition, the most significant genomic region obtained in this study was located at 48.61-48.84 Mb on GGA5. In this region, we have repeatedly identified four genes, in which YY1 (YY1 transcription factor) and WDR25 (WD repeat domain 25) have been shown to be related to oocytes and reproductive tissues, respectively, which implies that this region may be a candidate region underlying egg number traits. CONCLUSION: Our study utilized the genomic information from various chicken breeds or populations differed in the average annual egg number to understand the molecular genetic mechanisms involved in egg number traits. We identified a series of SNPs, candidate genes, or genomic regions that associated with egg number, which could help us in developing the egg production trait in chickens.

Genomic Regions Related to White/Black Tail Feather Color in Dwarf Chickens Identified Using a Genome-Wide Association Study.

Although the genetic foundation of chicken body feather color has been extensively explored, that of tail feather color remains poorly understood. In the present study, we used a synthetic chicken dwarf line (DW), derived from hybrids bred between a black tail chicken breed, Rhode Island Red (RIR), and a white tail breed, dwarf layer (DL), to investigate the genetic rules associated white/black tail color. Even though the body feathers are predominantly red, the DW line still comprises individuals with black or white tails after more than 10 generations of self-crossing and selection for the body feather color. We first performed four crosses using the DW chickens, including black-tailed males to females, reciprocal crosses between the black and white, and white males to females to elucidate the inheritance pattern of the white/black tail. We also performed a genome-wide association (GWA) analysis to determine the candidate genomic regions underlying the tail feather color using black tail chickens from the RIR and DW lines and white individuals from the DW line. In the crossing experiment, we found that (i) the white/black tail feather color is independent of body feather color; (ii) the phenotype is a simple autosomal trait; and (iii) the white is dominant to the black in the DW line. The GWA results showed that seven single-nucleotide polymorphisms (SNPs) on chromosome 24 were significantly correlated with tail feather color. The significant region (3.97-4.26 Mb) comprises nine known genes (NECTIN1, THY1, gga-mir-1466, USP2, C1QTNF5, RNF26, MCAM, CBL, and CCDC153) and five anonymous genes. This study revealed that the white/black tail feather trait is autosome-linked in DW chickens. Fourteen genes were found in the significant ~0.29 Mb genomic region, and some, especially MCAM, are suggested to play critical roles in the determination of white/black tail feather color. Our research is the first study on the genetics underlying tail feather color and could help further the understanding of feather pigmentation in chickens.

Genome-Wide Population Genetic Analysis of Commercial, Indigenous, Game, and Wild Chickens Using 600K SNP Microarray Data.

Following chicken domestication, diversified chicken breeds were developed by both natural and artificial selection, which led to the accumulation of abundant genetic and phenotypic variations, making chickens an ideal genetic research model. To better understand the genetic structure of chicken breeds under different selection pressures, we genotyped various chicken populations with specific selection targets, including indigenous, commercial, gamecock, and wild ancestral chickens, using the 600K SNP array. We analyzed the population structure, genetic relationships, run of homozygosity (ROH), effective population number (Ne), and other genetic parameters. The wild ancestral population, red junglefowl (RJF), possessed the highest diversity, in comparison with all other domesticated populations, which was supported by linkage disequilibrium decay (LD), effective population number, and ROH analyses. The gamecock breeds, which were subjected to stronger male-biased selection for fighting-related traits, also presented higher variation than the commercial and indigenous breeds. Admixture analysis also indicated that game breed is a relatively independent branch of Chinese local breeds. Following intense selection for reproductive and productive traits, the commercial lines showed the least diversity. We also observed that the European local chickens had lower genetic variation than the Chinese local breeds, which could be attributed to the shorter history of the European breed. ROH were present in a breed specific manner and 191 ROH island were detected on four groups (commercial, local, game and wild chickens). These ROH islands were involved in egg production, growth and silky feathers and other traits. Moreover, we estimated the effective sex ratio of these breeds to demonstrate the change in the ratio of the two sexes. We found that commercial chickens had a greater sex imbalance between females and males. The commercial lines showed the highest female-to-male ratios. Interestingly, RJF comprised a greater proportion of males than females. Our results show the population genetics of chickens under selection pressures, and can aid in the development of better conservation strategies for different chicken breeds.

Publisher Correction: Inheritance patterns of the transcriptome in hybrid chickens and their parents revealed by expression analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolution of cis- and trans-regulatory divergence in the chicken genome between two contrasting breeds analyzed using three tissue types at one-day-old.

BACKGROUND: Gene expression variation is a key underlying factor influencing phenotypic variation, and can occur via cis- or trans-regulation. To understand the role of cis- and trans-regulatory variation on population divergence in chicken, we developed reciprocal crosses of two chicken breeds, White Leghorn and Cornish Game, which exhibit major differences in body size and reproductive traits, and used them to determine the degree of cis versus trans variation in the brain, liver, and muscle tissue of male and female 1-day-old specimens. RESULTS: We provided an overview of how transcriptomes are regulated in hybrid progenies of two contrasting breeds based on allele specific expression analysis. Compared with cis-regulatory divergence, trans-acting genes were more extensive in the chicken genome. In addition, considerable compensatory cis- and trans-regulatory changes exist in the chicken genome. Most importantly, stronger purifying selection was observed on genes regulated by trans-variations than in genes regulated by the cis elements. CONCLUSIONS: We present a pipeline to explore allele-specific expression in hybrid progenies of inbred lines without a specific reference genome. Our research is the first study to describe the regulatory divergence between two contrasting breeds. The results suggest that artificial selection associated with domestication in chicken could have acted more on trans-regulatory divergence than on cis-regulatory divergence.

A genome-wide association study explores the genetic determinism of host resistance to Salmonella pullorum infection in chickens.

BACKGROUND: Salmonella infection is a serious concern in poultry farming because of its impact on both economic loss and human health. Chicks aged 20 days or less are extremely vulnerable to Salmonella pullorum (SP), which causes high mortality. Furthermore, an outbreak of SP infection can result in a considerable number of carriers that become potential transmitters, thus, threatening fellow chickens and offspring. In this study, we conducted a genome-wide association study (GWAS) to detect potential genomic loci and candidate genes associated with two disease-related traits: death and carrier state. METHODS: In total, 818 birds were phenotyped for death and carrier state traits through a SP challenge experiment, and genotyped by using a 600 K high-density single nucleotide polymorphism (SNP) array. A GWAS using a single-marker linear mixed model was performed with the GEMMA software. RNA-sequencing on spleen samples was carried out for further identification of candidate genes. RESULTS: We detected a region that was located between 33.48 and 34.03 Mb on chicken chromosome 4 and was significantly associated with death, with the most significant SNP (rs314483802) accounting for 11.73% of the phenotypic variation. Two candidate genes, FBXW7 and LRBA, were identified as the most promising genes involved in resistance to SP. The expression levels of FBXW7 and LRBA were significantly downregulated after SP infection, which suggests that they may have a role in controlling SP infections. Two other significant loci and related genes (TRAF3 and gga-mir-489) were associated with carrier state, which indicates a different polygenic determinism compared with that of death. In addition, genomic inbreeding coefficients showed no correlation with resistance to SP within each breed in our study. CONCLUSIONS: The results of this GWAS with a carefully organized Salmonella challenge experiment represent an important milestone in understanding the genetics of infectious disease resistance, offer a theoretical basis for breeding SP-resistant chicken lines using marker-assisted selection, and provide new information for salmonellosis research in humans and other animals.

Genome-Wide Single-Nucleotide Polymorphism Data Unveil Admixture of Chinese Indigenous Chicken Breeds with Commercial Breeds.

The conservation and development of chicken have received considerable attention, but the admixture history of chicken breeds, especially Chinese indigenous breeds, has been poorly demonstrated. In this study, we aimed to evaluate the genetic diversity and population structure of eight chicken breeds (including conserved chicken breeds) from different geographic origin and to identify admixture within these breeds using a 600-K single-nucleotide polymorphism panel for genotyping. Using the genotype of 580,961 single-nucleotide polymorphism markers scored in 1,200 animals, we evaluated the genetic diversity (heterozygosity and proportion of polymorphic markers), linkage disequilibrium decay, population structure (principal component analysis and neighbor-joining tree), genetic differentiation (FST and genetic distance), and migration events (TreeMix and f-statistics) of the eight domesticated chicken breeds. The results of population analytical methods revealed patterns of hybridization that occurred after divergence in Tibetan chicken. We argue that chicken migration and admixture, followed by trade, have been important forces in shaping the genomic variation in modern Chinese chicken. Moreover, isolation by distance might play a critical role in shaping the genomic variation within Eurasia continent chicken breeds. Moreover, genetic information provided in this study is valuable resources for production applications (genomic prediction, and breeding strategy) and scientific research (genetic basis detection, studying evolution, or domestication).

Feather colour affects the aggressive behaviour of chickens with the same genotype on the dominant white (I) locus.

Aggression in chickens is a serious economic and animal welfare issue in poultry farming. Pigmentation traits have been documented to be associated with animal behaviour. Chicken pecking behaviour has been found to be related to feather colour, with premelanosome protein 17 (PMEL17) being one of the candidate genes. In the present study, we performed a genotypic and phenotypic association analysis between chicken plumage colour (red and white) and aggressive behaviour in an F1 hybrid group generated by crossing the autosomal dominant white-feathered breed White Leghorn (WL) and the red-feathered breed Rhode Island Red (RIR). In genetic theory, all the progeny should have white feathers because WL is homozygous autosomal dominant for white feathers. However, we found a few red-feathered female chickens. We compared the aggressiveness between the red and white females to determine whether the feather colour alone affected the behaviour, given that the genetic background should be the same except for feather colour. The aggressiveness was recorded 5 days after sexual maturity at 26 weeks. Generally, white plumage hens showed significantly higher aggressiveness compared to the red ones in chasing, attacking, pecking, and threatening behaviour traits. We assessed three candidate feather colour genes-PMEL17, solute carrier family 45 member 2 (SLC45A2), and SRY-box 10 (SOX10)-to determine the genetic basis for the red and white feather colour in our hybrid population; there was no association between the three loci and feather colour. The distinct behavioural findings observed herein provide clues to the mechanisms underlying the association between phenotype and behaviour in chickens. We suggest that mixing red and white chickens together might reduce the occurrence of aggressive behaviours.

Inheritance patterns of the transcriptome in hybrid chickens and their parents revealed by expression analysis.

Although many phenotypic traits of chickens have been well documented, the genetic patterns of gene expression levels in chickens remain to be determined. In the present study, we crossed two chicken breeds, White Leghorn (WL) and Cornish (Cor), which have been selected for egg and meat production, respectively, for a few hundred years. We evaluated transcriptome abundance in the brain, muscle, and liver from the day-old progenies of pure-bred WL and Cor, and the hybrids of these two breeds, by RNA-Seq in order to determine the inheritance patterns of gene expression. Comparison among expression levels in the different groups revealed that most of the genes showed conserved expression patterns in all three examined tissues and that brain had the highest number of conserved genes, which indicates that conserved genes are predominantly important compared to others. On the basis of allelic expression analysis, in addition to the conserved genes, we identified the extensive presence of additive, dominant (Cor dominant and WL dominant), over-dominant, and under-dominant genes in all three tissues in hybrids. Our study is the first to provide an overview of inheritance patterns of the transcriptome in layers and broilers, and we also provide insights into the genetics of chickens at the gene expression level.

The Genetic Architecture of Early Body Temperature and Its Correlation With Salmonella Pullorum Resistance in Three Chicken Breeds.

New-born chicks are vulnerable to bacterial infections and not good at regulating body temperature. There is a close relationship between thermal regulation and immunity, however, the underlying mechanism is not well understood. Salmonella Pullorum (SP) is a major concern in developing countries and causes significant economic losses in poultry industry. Early body temperature (EBT) has previously shown to be correlated with host immunity and resistance to pullorum disease. In this study, we challenged three independent chick populations (Beijing You, Dwarf and Rhode Island Red) with SP at 4 days of age, and rectal temperature was measured before and after the SP attack from 2 to 7 days of age. Host defense to SP was evaluated by survival and spleen SP carrier status. The results showed that chicks with higher EBT before SP infection tend to have higher resistance to later SP attack in two populations (Dwarf and Beijing You). The association between EBT before SP attack and SP resistance was non-significant in Rohde Island Red population (P = 0.06), but the trend was consistent with the other two populations. We also found low to moderate heritability in all three populations for EBT before and after the SP attack ranging from 0.14 to 0.20. Genome-wide association studies identified several genomic regions and biological pathways determining EBT before SP attack, which provides candidate functional genes of this trait. Our results reveal the genetic determination of EBT, and the relationship between EBT and SP resistance, providing an alternative strategy for improving SP resistant activities in chicken.

Evaluation of genetic resistance to Salmonella Pullorum in three chicken lines.

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.

Genome-wide association study revealed genomic regions related to white/red earlobe color trait in the Rhode Island Red chickens.

BACKGROUND: Earlobe color is a naturally and artificially selected trait in chicken. As a head furnishing trait, it has been selected as a breed characteristic. Research has demonstrated that white/red earlobe color was related to at least three loci and sex-linked. However, there has been little work to date to identify the specific genomic regions and genes response to earlobe color in Rhode Island Red chickens. Currently, it is possible to identify the genomic regions responsible for white/red earlobe in Rhode Island Red chicken to eliminate this gap in knowledge by using genome-wide association (GWA) analysis. RESULTS: In the present study, genome-wide association (GWA) analysis was conducted to explore the candidate genomic regions response to chicken earlobe color phenotype. Hens with red dominant and white dominant earlobe was used for case-control analysis by Illumina 600 K SNP arrays. The GWA results showed that 2.38 Mb genomic region (50.13 to 52.51 Mb) with 282 SNPs on chromosome Z were significantly correlated to earlobe color, including sixteen known genes and seven anonymous genes. The sixteen genes were PAM, SLCO4C1, ST8SIA4, FAM174A, CHD1, RGMB, RIOK2, LIX1, LNPEP, SHB, RNF38, TRIM14, NANS, CLTA, GNE, and CPLX1. CONCLUSIONS: The study has revealed the white/red earlobe trait is polygenic and sex-linked in Rhode Island Red chickens. In the genome significant ~2.38 Mb region, twenty-three genes were found and some of them could play critical roles in the formation of white/red earlobe color, especially gene SLCO4C1. Taken together, the candidate genes findings herein can help elucidate the genomic architecture of response to white/red earlobe and provide a new insight on mechanisms underlying earlobe color in Rhode Island Red chickens and other breeds.

Parallel Evolution of Polydactyly Traits in Chinese and European Chickens.

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.