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Abstract Background Chicken is one of the most numerous and widely distributed species around the world, and many studies support the multiple ancestral origins of domestic chickens. The research regarding the yellow skin phenotype in domestic chickens (regulated by BCO2) likely originating from the grey junglefowl serves as crucial evidence for demonstrating the multiple origins of chickens. However, beyond the BCO2 gene region, much remains unknown about the introgression from the grey junglefowl into domestic chickens. Therefore, in this study, based on whole-genome data of 149 samples including 4 species of wild junglefowls and 13 local domestic chicken breeds, we explored the introgression events from the grey junglefowl to domestic chickens. Results We successfully detected introgression regions besides BCO2, including two associated with growth trait (IGFBP2 and TKT), one associated with angiogenesis (TIMP3) and two members of the heat shock protein family (HSPB2 and CRYAB). Our findings suggest that the introgression from the grey junglefowl may impact the growth performance of chickens. Furthermore, we revealed introgression events from grey junglefowl at the BCO2 region in multiple domestic chicken breeds, indicating a phenomenon where the yellow skin phenotype likely underwent strong selection and was retained. Additionally, our haplotype analysis shed light on BCO2 introgression event from different sources of grey junglefowl into domestic chickens, possibly suggesting multiple genetic flows between the grey junglefowl and domestic chickens. Conclusions In summary, our findings provide evidences of the grey junglefowl contributing to the genetic diversity of domestic chickens, laying the foundation for a deeper understanding of the genetic composition within domestic chickens, and offering new perspectives on the impact of introgression on domestic chickens.

ABSTRACT: In the late growth stage of commercial Pekin ducks, a significant increase in feed intake and a decline in body weight gain have been observed, leading to impaired feed conversion efficiency. To address this issue, we investigated alterations in production performance, blood biochemical indices, ileum tissue architecture, and microbial community structure in Pekin ducks. The primary objective was to provide robust data supporting the improvement of meat duck production efficiency during the late growth stage (28–42-days-old). Forty 28-day-old Pekin ducks were randomly assigned to 8 replicates, with five ducks per replicate. The rearing period lasted 14 days, with feed and water provided ad libitum. Our findings indicated a significant increase in Pekin duck body and heart weights with advancing age (P < 0.05). Moreover, serum antioxidant enzyme and high-density lipoprotein concentrations significantly increased, whereas triglyceride levels decreased (P < 0.05). Notably, the height of the ileal villi was significantly reduced (P < 0.05). The microbial community structure of the ileum exhibited significant changes as ducks aged, accompanied by a substantial increase in microbial flora diversity, particularly with the formation of more tightly connected microbial network modules. Time-dependent enrichment was observed in microbial gene functions related to energy metabolism pathways. At the genus level, Sphingomonas and Subdoligranulum have emerged as crucial players in microbial differential functional pathways and network formation. These bacteria likely serve as the key driving factors in the dynamic microbial changes that occur in Pekin ducks over time. Overall, our findings suggest a potential decline in the absorption function of the small intestine and fat deposition performance of Pekin ducks during later growth stages, which may be attributed to the maturation and proliferation of the gut microbial community.

Abstract Background Avian pathogenic E. coli (APEC) can cause localized or systemic infections, collectively known as avian colibacillosis, resulting in huge economic losses to poultry industry globally per year. In addition, increasing evidence indicates that long non-coding RNAs (lncRNAs) play a critical role in regulating host inflammation in response to bacterial infection. However, the role of lncRNAs in the host response to APEC infection remains unclear. Results Here, we found 816 differentially expressed (DE) lncRNAs and 1,798 DE mRNAs in APEC infected chicken macrophages by RNAseq. The identified DE lncRNA-mRNAs were involved in Toll like receptor signaling pathway, VEGF signaling pathway, fatty acid metabolism, phosphatidylinositol signaling system, and other types of O-glycan biosynthesis. Furthermore, we found the novel lncRNA TCONS_00007391 as an important immune regulator in APEC infection was able to regulate the inflammatory response by directly targeting CD86. Conclusion These findings provided a better understanding of host response to APEC infection and also offered the potential drug targets for therapy development against APEC infection.

ABSTRACT: The bird beak is mainly functioned as feeding and attacking, and its shape has extremely important significance for survival and reproduction. In chickens, since beak shape could lead to some disadvantages including pecking and waste of feed, it is important to understand the inheritance of chicken beak shape. In the present study, we firstly established 4 indicators to describe the chicken beak shapes, including upper beak length (UL), lower beak length (LL), distance between upper and lower beak tips (DB) and upper beak curvature (BC). And then, we measured the 4 beak shape indicators as well as some production traits including body weight (BW), shank length (SL), egg weight (EW), eggshell strength (ES) of a layer breed, Rhode Island Red (RIR), in order to estimate genetic parameters of chicken beak shape. The heritabilities of UL and LL were 0.41 and 0.37, and the heritabilities of DB and BC were 0.22 and 0.21, indicating that beak shape was a highly or mediumly heritable. There were significant positive genetic and phenotypic correlations among UL, LL, and DB. And UL was positively correlated with body weight (BW18) and shank length (SL18) at 18 weeks of age in genetics, and DB was positively correlated with BC in terms of genetics and phenotype. We also found that layers of chicken cages played a role on beak shape, which could be attributed to the difference of lightness in different cage layers. By a genome-wide association study (GWAS) for the chicken UL, we identified 9 significant candidate genes associated with UL in RIR. For the variants with low minor allele frequencies (MAF

ABSTRACT: Plumage color is a characteristic trait of ducks that originates as a result of natural and artificial selection. As a conspicuous phenotypic feature, it is a breed characteristic. Previous studies have identified some genes associated with the formation of black and white plumage in ducks. However, studies on the genetic basis underlying the red plumage phenotype in ducks are limited. Here, genome-wide association analysis (GWAS) and selection signal detection (Fst, θπ ratio, and cross-population composite likelihood ratio [XP-CLR]) were conducted to identify candidate regions and genes underlying duck plumage color phenotype. Selection signal detection revealed 29 overlapping genes (including ENPP1 and ULK1) significantly associated with red plumage color in Ji'an Red ducks. ENSAPLG00000012679, ESRRG, and SPATA5 were identified as candidate genes associated with red plumage using GWAS. Selection signal detection revealed that 19 overlapping genes (including GMDS, PDIA6, and ODC1) significantly correlated with light brown plumage in Brown Tsaiya ducks. GWAS to narrow down the significant regions further revealed nine candidate genes (AKT1, ATP6V1C2, GMDS, LRP4, MAML3, PDIA6, PLD5, TMEM63B, and TSPAN8). Notably, in Brown Tsaiya ducks, GMDS, ODC1, and PDIA6 exhibit significantly differentiated allele frequencies among other feather-colored ducks, while in Ji'an Red ducks, ENSAPLG00000012679 has different allele frequency distributions compared with that in other feather-colored ducks. This study offers new insights into the variation and selection of the red plumage phenotype using GWAS and selective signals.

ABSTRACT: As a Chinese local chicken breed, Hongshan chickens have 2 kinds of tail feather phenotypes, normal and taillessness. Our previous studies showed that taillessness was a sex-linked dominant trait. Abnormal development of the tail vertebrae could be explained this phenomenon in some chicken breeds. However, the number of caudal vertebrae in rumpless Hongshan chickens was normal, so rumplessness in Hongshan chicken was not related to the development of the caudal vertebrae. Afterwards, we found that rumplessness in Hongshan was due to abnormal development of tail feather rather than abnormal development of caudal vertebrae. In order to understand the genetic foundation of the rumplessness of Hongshan chickens, we compared and reanalyzed 2 sets of data in normal and rumpless Hongshan chickens from our previous studies. By joint analysis of genome-wide selection signature analysis and genome-wide association approach, we found that 1 overlapping gene (EDIL3) and 16 peak genes (ENSGALG00000051843, ENSGALG00000053498, ENSGALG00000054800, KIF27, PTPRD, ENSGALG00000047579, ENSGALG00000041052, ARHGEF28, CAMK4, SERINC5, ENSGALG00000050776, ERCC8, MCC, ADAMTS19, ENSGALG00000053322, CHRNA8) located on the Z chromosome was associated with the rumpless trait. The results of this study furtherly revealed the molecular mechanism of the rumpless trait in Hongshan chickens, and identified the candidate genes associated with this trait. Our results will help to improve the shape of chicken tail feathers and to rise individual economic value in some specific market in China.

ABSTRACT: The age of first egg (AFE) in chicken can affect early and even life-time egg production performance to some extent, and therefore is an important economic trait that affects production efficiency. To better understand the genetic patterns of AFE and other production traits including body weight at first egg (BWA), first egg weight (FEW), and total egg number from AFE to 58 wk of age (total-EN), we recorded the production performance of 2 widely used layer breeds, white leghorn (WL) and Rhode Island Red (RIR) and estimated genetic parameters based on pedigree and production data. The results showed that the heritability of AFE in both breeds ranged from 0.4 to 0.6, and AFE showed strong positive genetic and phenotypic correlations to BWA as well as FEW, while showing strong negative genetic and phenotypic correlations with total-EN. Furtherly, by genome-wide association analysis study (GWAS), we identified 12 and 26 significant SNPs to be related to AFE in the 2-layer breeds, respectively. A total of 18 genes were identified that could affect AFE based on the significant SNP annotations obtained, but there were no gene overlapped in the 2 breeds indicating the genetic foundation of AFE could differ from breed to breed. Our results provided a deeper understanding of genetic patterns and molecular basement of AFE in different breeds and could help in the selection of egg production traits.

Abstract Background Eggshell speckle phenotype is an important trait in poultry production because they affect eggshell quality. However, the genetic architecture of speckled eggshells remains unclear. In this study, we determined the heritability of eggshell speckles and conducted a genome-wide association study (GWAS) on purebred Rhode Island Red (RIR) hens at 28 weeks to detect potential genomic loci and candidate genes associated with eggshell speckles. Results The heritability of eggshell speckles was 0.35 at 28 weeks, and the speckle level is not related to other eggshell quality traits in terms of phenotypic correlation. We detected 311 SNPs (6 significantly, and 305 suggestively associated) and 39 candidate genes associated with eggshell speckles. Based on the pathway analysis, the 39 candidate genes were mainly involved in alpha-linolenic acid metabolism, linoleic acid metabolism, ether lipid metabolism, GnRH signaling pathway, vascular smooth muscle contraction, and MAPK signaling pathway. Ultimately, ten genes, LOC423226, SPTBN5, EHD4, LOC77155, TYRO3, ITPKA, DLL4, PLA2G4B, PLA2G4EL5, and PLA2G4EL6 were considered the most promising genes associated with eggshell speckles that were implicated in immunoregulation, calcium transport, and phospholipid metabolism, while its function in laying hens requires further studies. Conclusions This study provides new insights into understanding the genetic basis of eggshell speckles and has practical application value for the genetic improvement of eggshell quality.

Spurs, which mainly appear in roosters, are protrusions near the tarsometatarsus on both sides of the calves of chickens, and are connected to the tarsometatarsus by a bony core. As a male-biased morphological characteristic, the diameter and length of spurs vary significantly between different individuals, mainly related to genetics and age. As a specific behavior of hens, egg-laying also varies greatly between individuals in terms of traits such as age at first egg (AFE), egg weight (EW), and so on. At present, there are few studies on chicken spurs. In this study, we investigated the inheritance pattern of the spur trait in roosters with different phenotypes and the correlations between spur length, body weight at 18 weeks of age (BW18), shank length at 18 weeks of age (SL18), and the egg-laying trait in hens (both hens and roosters were from the same population and were grouped according to their family). These traits related to egg production included AFE, body weight at first egg (BWA), and first egg weight (FEW). We estimated genetic parameters based on pedigree and phenotype data, and used variance analysis to calculate broad-sense heritability for correcting the parameter estimation results. The results showed that the heritability of male left and right spurs ranged from 0.6 to 0.7. There were significant positive correlations between left and right spur length, BW18, SL18, and BWA, as well as between left and right spur length and AFE. We selected 35 males with the longest spurs and 35 males with the shortest spurs in the population, and pooled them into two sets to obtain the pooled genome sequencing data. After genome-wide association and genome divergency analysis by FST, allele frequency differences (AFDs), and XPEHH methods, we identified 7 overlapping genes (CENPE, FAT1, FAM149A, MANBA, NFKB1, SORBS2, UBE2D3) and 14 peak genes (SAMD12, TSPAN5, ENSGALG00000050071, ENSGALG00000053133, ENSGALG00000050348, CNTN5, TRPC6, ENSGALG00000047655,TMSB4X, LIX1, CKB, NEBL, PRTFDC1, MLLT10) related to left and right spur length through genome-wide selection signature analysis and a genome-wide association approach. Our results identified candidate genes associated with chicken spurs, which helps to understand the genetic mechanism of this trait and carry out subsequent research around it.

ABSTRACT: Cockfighting is popular worldwide, dating back to 2,800 BC. Primarily, 5 modern Chinese gamecock breeds exist, located in the northeast (Luxi and Henan), west (Turpan), south (Xishuangbanna), and southeast (Zhangzhou) of China. However, whether Chinese gamecocks were derived from a single origin or multiple origins remains controversial. Therefore, this study used next-generation resequencing data to elucidate the origin of Chinese gamecocks by constructing genome-wide and SRY-box transcription factor 5 (SOX5) gene phylogenetic trees. Data from 161 chickens from 27 breeds, including 9 gamecock breeds, were included. Before constructing the SOX5 gene tree, we validated that the pea-comb phenotype mutation in all gamecock breeds was attributed to copy number variation in intron 1 of the SOX5 gene, as previously reported. The specific region was chr1: 65,838,000 to 65,846,000. The phylogenetic tree results suggested that Zhangzhou and Xishuangbanna gamecocks have a monophyletic origin, while Luxi, Henan, and Turpan gamecocks have a common ancestor. Our study provides genome-wide evidence that Chinese gamecocks have multiple origins and advances the understanding of the genetic mechanisms of the pea-comb characteristic.

ABSTRACT: Body weight of chicken is a typical quantitative trait, which shows phenotypic variations due to selective breeding. Despite some QTL loci have been obtained, the body weight of native chicken breeds in different geographic regions varies greatly, its genetic basis remains unresolved questions. To address this issue, we analyzed 117 Chinese indigenous chickens from 10 breeds (Huiyang Bearded, Xinhua, Hotan Black, Baicheng You, Liyang, Yunyang Da, Jining Bairi, Lindian, Beijing You, Tibetan). We applied fixation index (FST) analysis to find selected genomic regions and genes associated with body weight traits. Our study suggests that NELL1, XYLT1, and NCAPG/LCORL genes are strongly selected in the body weight trait of Chinese indigenous chicken breeds. In addition, the IL1RAPL1 gene was strongly selected in large body weight chickens, while the PCDH17 and CADM2 genes were strongly selected in small body weight chickens. This result suggests that the patterns of genetic variation of native chicken and commercial chicken, and/or distinct local chicken breeds may follow different evolutionary mechanisms.

Abstract Background Geese are among the most important poultry species in the world. The current generally accepted hypothesis is that the European domestic geese originated from greylag geese (Anser anser), and Chinese domestic geese have two origins, most of which originated from swan geese (Anser cygnoides), and the Yili goose originated from greylag geese. To explain the origin and demographic history of geese, we selected 14 goose breeds from Europe and China and wild populations of swan and greylag geese, and whole genome sequencing data were obtained for 74 samples. Results Population structure analysis and phylogenetic trees showed that the wild ancestor of Chinese domestic geese, except for Yili, is the swan geese, and the wild ancestor of Chinese Yili and European domestic geese is greylag geese. Analysis of the demographic history suggests that the domestication of Chinese geese occurred ~ 3499 years ago and that of the European geese occurred ~ 7552 years ago. Furthermore, gene flow was observed between domestic geese and their wild ancestors. Analysis of introgression showed that Yili geese had been introgressed by Chinese domestic geese, and the body size of Yili geese may be influenced by introgression events of some growth-related genes, including IGF-1. Conclusions Our study provides evidence for the origin of geese at the genome-wide level and advances the understanding of the history of goose domestication and the traits affected by introgression events.

Abstract Background The quality of poultry eggshells is closely related to the profitability of egg production. Eggshell speckles reflect an important quality trait that influences egg appearance and customer preference. However, the mechanism of speckle formation remains poorly understood. In this study, we systematically compared serum immune and antioxidant indices of hens laying speckled and normal eggs. Transcriptome and methylome analyses were used to elucidate the mechanism of eggshell speckle formation. Results The results showed that seven differentially expressed genes (DEGs) were identified between the normal and speckle groups. Gene set enrichment analysis (GSEA) revealed that the expressed genes were mainly enriched in the calcium signaling pathway, focal adhesion, and MAPK signaling pathway. Additionally, 282 differentially methylated genes (DMGs) were detected, of which 15 genes were associated with aging, including ARNTL, CAV1, and GCLC. Pathway analysis showed that the DMGs were associated with T cell-mediated immunity, response to oxidative stress, and cellular response to DNA damage stimulus. Integrative analysis of transcriptome and DNA methylation data identified BFSP2 as the only overlapping gene, which was expressed at low levels and hypomethylated in the speckle group. Conclusions Overall, these results indicate that aging- and immune-related genes and pathways play a crucial role in the formation of speckled eggshells, providing useful information for improving eggshell quality.

ABSTRACT: Genomic admixture is a widespread phenomenon among domestic animal breeds, including chickens. However, reports on admixture within Chinese gamecocks or other indigenous chickens are limited. This study focuses on the population genetic structure and admixture of 5 Chinese gamecock breeds and the admixture with 9 other indigenous Chinese chicken breeds. Our results showed that Turpan and Henan gamecocks were grouped into one cluster, whereas Luxi, Zhangzhou, and Xishuangbanna gamecocks were grouped into the other cluster. Gene flow occurred between Xishuangbanna and Turpan and Turpan and Luxi gamecocks. Simultaneously, gene flow was observed between gamecocks and indigenous chickens, such as Xishuangbanna and Wenchang. Ancestral component analysis indicated that modern domestic chickens in southern China played an important role in the history of the domestication of modern Chinese gamecock. Our study will be helpful in better understanding the domestication and evolution of Chinese gamecock.

ABSTRACT: High dropping moisture (DM) in poultry production has deleterious effects on the environment, feeding cost, and public health of people and animals. To explore the contributing genetic components, we classified DM of 67-wk-old Rhode Island Red (RIR) hens at 4 different levels and evaluated the underlying genetic heritability. We found the heritability of DM to be 0.219, indicating a moderately heritable trait. We then selected chickens with the highest and lowest DM levels. Using transcriptome, we only detected 12 differentially expressed genes (DEGs) between these 2 groups from the spleen, and 1,507 DEGs from intestinal tissues (jejunum and cecum). The low number of DEGs observed in the spleen suggests that differing moisture levels are not attributed to pathogenic infection. Fourteen of the intestinal high expressed genes are associated with water-salt metabolism (WSM). We also investigated the gut microbial composition by 16S rRNA gene amplicon sequencing. Six different microbial operational taxonomic units (OTUs) (Cetobacterium, Sterolibacterium, Elusimicrobium, Roseburia, Faecalicoccus, and Megamonas) between the 2 groups from jejunum and cecum are potentially biomarkers related to DM levels. Our results identify a genetic component to chicken DM, and can guide breeding strategies.

Genes with sex-biased expression are thought to underlie sexually dimorphic phenotypes and are therefore subject to different selection pressures in males and females. Many authors have proposed that sexual conflict leads to the evolution of sex-biased expression, which allows males and females to reach separate phenotypic and fitness optima. The selection pressures associated with domestication may cause changes in population architectures and mating systems, which in turn can alter their direction and strength. We compared sex-biased expression and genetic signatures in wild and domestic ducks (Anas platyrhynchos), and observed changes of sexual selection and identified the genomic divergence affected by selection forces. The extent of sex-biased expression in both sexes is positively correlated with the level of both dN/dS and nucleotide diversity. This observed changing pattern may mainly be owing to relaxed genetic constraints. We also demonstrate a clear link between domestication and sex-biased evolutionary rate in a comparative framework. Decreased polymorphism and evolutionary rate in domesticated populations generally matched life-history phenotypes known to experience artificial selection. Taken together, our work suggests the important implications of domestication in sex-biased evolution and the roles of artificial selection and sexual selection for shaping the diversity and evolutionary rate of the genome.

Driven by natural and artificial selection, the domestic Huoyan geese from Northern China have gradually generated specific phenotypes and climatic adaptations. To understand the genetic basis of the two specific phenotypes that are sex linked, including upper eyelid coloboma and gosling feather color, as well as the climatic adaptations of the Huoyan goose, which can contribute to the artificial selection and breeding of geese. We selected Huoyan geese and nine Southern Chinese goose breeds and identified their divergence on the genomic level. Using selective sweep analysis, we found that PTPRM on chromosome Z influences the upper eyelid coloboma phenotype of the Huoyan goose, and TYRP1 is a plausible candidate gene for the Huoyan gosling feather color. We obtained a number of genes related to cold adaptation in Huoyan geese, mainly involved in physiological functions such as metabolism, angiogenesis contraction and circulatory system, apoptosis, immunity, stress, and neural system. The most interesting candidates for cold adaptation are PIP5K1B and NMNAT3 that are associated with energy metabolism and stress. We also obtained some genes related to heat adaptation, including AGTPBP1, associated with neurology; GDA, associated with skin pigmentation; and NAA35, associated with apoptosis. These findings deepen our understanding of the genetics of specific phenotypes and climate adaptation in local geese and provide insights for the selection of goose breeds.

Abstract Background Receptor interacting serine/threonine kinase 2 (RIP2), ubiquitous in many tissue/cell types, is the key regulator of immune and inflammatory responses for many diseases, including avian pathogenic E. coli (APEC), which causes a wide variety of localized or systemic infections. However, the molecular mechanisms by which RIP2 drives its transcriptional program to affect immune and inflammatory response upon APEC infection remains poorly understood. Results In this study, RNA-seq and bioinformatics analyses were used to detect gene expression and new direct/indirect RIP2 targets in the treatments of wild type HD11 cells (WT), RIP2 knockdown cells (shRIP2), APEC stimulation cells (APEC), and RIP2 knockdown cells combined with APEC infection (shRIP2 + APEC). The results revealed that a total of 4691 and 2605 differentially expressed genes (DEGs) were screened in shRIP2 + APEC vs. APEC and shRIP2 vs. WT, respectively. Functional annotation analysis showed that apoptosis, MAPK, p53, Toll-like receptor, and Nod-like receptor signaling pathways were involved in APEC-induced RIP2 knockdown HD11 cells. By analyzing the enriched pathway and gene networks, we identified that several DEGs, including HSP90AB1, BID, and CASP9 were targeted by RIP2 upon APEC infection. Conclusion As a whole, this study can not only provide data support for constructing gene networks of RIP2 knockdown with APEC challenge but also provide new ideas for improving the immune and inflammatory response.

ABSTRACT: Speckles are common on the brown eggshells of chicken eggs, especially for aged hens. They are important as they affect the consumer preference and economic value of eggs. The cause of speckles in eggshells is still unclear. In this study, we verified the difference of eggshell quality between speckled eggs and normal eggs. Structural characteristics of speckled eggshells were investigated using a scanning electron microscope. Results showed no significant difference in the eggshell quality between normal eggs and speckled eggs, except for a lower eggshell color–L value in the latter. More materials deposited between vertical crystal layer and cuticle layer in the speckled shell region, leading to the thicker eggshell than adjacent normal area. The relative content of protoporphyrin IX was significantly higher in the speckled area than in the adjacent normal area of the eggshells. In addition, there was no significant differences in productive and reproductive performance between hens that laid normal eggs or speckled eggs, except for a lower hatchability of the speckled eggs. In conclusion, we infer that the uneven distribution of eggshell pigment protoporphyrin IX leads to the formation of speckled eggs. This is the first study to establish the characteristics and causes of speckled eggshells. Moreover, this study provides novel insights into external egg quality and a foundation for the in-depth study of speckled eggshells.

Chicken plumage color is an inheritable phenotype that was naturally and artificially selected for during domestication. The Baicheng You chicken is an indigenous Chinese chicken breed presenting three main feather colors, lavender, black, and yellow plumages. To explore the genetic mechanisms underlying the pigmentation in Baicheng You chickens, we re-sequenced the whole genome of Baicheng You chicken with the three plumage colors. By analyzing the divergent regions of the genome among the chickens with different feather colors, we identified some candidate genomic regions associated with the feather colors in Baicheng You chickens. We found that EGR1, MLPH, RAB17, SOX5, and GRM5 genes were the potential genes for black, lavender, and yellow feathers. MLPH, GRM5, and SOX5 genes have been found to be related to plumage colors in birds. Our results showed that EGR1 is a most plausible candidate gene for black plumage, RAB17, MLPH, and SOX5 for lavender plumage, and GRM5 for yellow plumage in Baicheng You chicken.

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.

Abstract Background Domestication alters several phenotypic, neurological, and physiological traits in domestic animals compared to those in their wild ancestors. Domestic ducks originated from mallards, and some studies have shown that spot-billed ducks may have also made minor genetic contributions to domestication. Compared with the two ancestral species, domestic ducks generally differ in body size and bone morphology. In this study, we performed both genomic and transcriptomic analyses to identify candidate genes for elucidating the genetic mechanisms underlying phenotypic variation. Methods In this study, the duck genome data from eight domestic breeds and two wild species were collected to study the genetic changes during domestication. And the transcriptome data of different tissues from wild ducks and seven domestic ducks were used to reveal the expression difference between wild and domestic ducks. Results Using fixation index (Fst) algorithm and transcriptome data, we found that the genes related to skeletal development had high Fst values in wild and domestic breeds, and the differentially expressed genes were mainly enriched in the ossification pathway. Our data strongly suggest that the skeletal systems of domestic ducks were changed to adapt to artificial selection for larger sizes. In addition, by combining the genome and transcriptome data, we found that some Fst candidate genes exhibited different expression patterns, and these genes were found to be involved in digestive, immune, and metabolic functions. Conclusions A wide range of phenotypic differences exists between domestic and wild ducks. Through both genome and transcriptome analyses, we found that genes related to the skeletal system in domestic ducks were strongly selected. Our findings provide new insight into duck domestication and selection effects during the domestication.

Abstract 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.

Feather damage (FD) in laying hens is an economic and welfare issue globally. FD can be influenced by environmental, genetic, and nutrient factors. It is worth understanding the inheritance patterns and influencers of FD in layers for further improvement of feather quality. In the present study, tail and neck feather coverage of White Leghorn (WL) and Rhode Island Red (RIR) hens were recorded to evaluate FD. The genetic parameters of FD and their impact on the production performance were estimated. The heritability of the tail and neck FD for White Leghorn is 0.22 and 0.09, respectively, and the genetic correlation between them is 0.40. The heritability of neck FD of Rhode Island Red is 0.27. FD had a limited influence on egg production. Collectively, FD is influenced by many factors, including genetic factors, appropriate breeding strategies, and improved feeding environments, which are necessary to reduce FD in laying hens.

Plumage color is an artificially and naturally selected trait in domestic ducks. Black, white, and spotty are the main feather colors in domestic ducks. Previous studies have shown that black plumage color is caused by MC1R, and white plumage color is caused by MITF. We performed a genome-wide association study (GWAS) to identify candidate genes associated with white, black, and spotty plumage in ducks. Two non-synonymous SNPs in MC1R (c.52G>A and c.376G>A) were significantly related to duck black plumage, and three SNPs in MITF (chr13:15411658A>G, chr13:15412570T>C and chr13:15412592C>G) were associated with white plumage. Additionally, we also identified the epistatic interactions between causing loci. Some ducks with white plumage carry the c.52G>A and c.376G>A in MC1R, which also compensated for black and spotty plumage color phenotypes, suggesting that MC1R and MITF have an epistatic effect. The MITF locus was supposed to be an upstream gene to MC1R underlying the white, black, and spotty colors. Although the specific mechanism remains to be further clarified, these findings support the importance of epistasis in plumage color variation in ducks.

Selection pressures driven by natural causes or human interference are key factors causing genome variants and signatures of selection in specific regions of the genome. Gamecocks were bred for cockfighting, presenting pea-combs, larger body sizes, stronger limbs, and higher levels of aggression than other chickens. In this study, we aimed to explore the genomic differences between Chinese gamecocks and commercial, indigenous, foreign, and cultivated breeds by detecting the regions or sites under natural or artificial selection using genome-wide association studies (GWAS), genome-wide selective sweeps based on the genetic differentiation index (FST), and transcriptome analyses. Ten genes were identified using GWAS and FST: gga-mir-6608-1, SOX5, DGKB, ISPD, IGF2BP1, AGMO, MEOX2, GIP, DLG5, and KCNMA1. The ten candidate genes were mainly associated with muscle and skeletal development, glucose metabolism, and the pea-comb phenotype. Enrichment analysis results showed that the differentially expressed genes between the Luxi (LX) gamecock and Rhode Island Red (RIR) chicken were mainly related to muscle development and neuroactive-related pathways. This study will help to understand the genetic basis and evolution of Chinese gamecocks and support the further use of gamecocks as an excellent breeding material from a genetic perspective.

Epigenetic modifications play an important role in regulating animal adaptation to external stress. To explore how DNA methylation regulates the expression levels of related genes during forced molting (FM) of laying hens, the hypothalamus and ovary tissues were analyzed at five periods using Whole-Genome Bisulfite Sequencing. The results show that methylation levels fluctuated differently in the exon, intron, 5′UTR, 3′UTR, promoter, and intergenic regions of the genome during FM. In addition, 16 differentially methylated genes (DMGs) regulating cell aging, immunity, and development were identified in the two reversible processes of starvation and redevelopment during FM. Comparing DMGs with differentially expressed genes (DEGs) obtained in the same periods, five hypermethylated DMGs (DSTYK, NKTR, SMOC1, SCAMP3, and ATOH8) that inhibited the expression of DEGs were found. Therefore, DMGs epigenetically modify the DEGs during the FM process of chickens, leading to the rapid closure and restart of their reproductive function and a re-increase in the egg-laying rate. Therefore, this study further confirmed that epigenetic modifications could regulate gene expression during FM and provides theoretical support for the subsequent optimization of FM technology.

Bone health is particularly important for high-yielding commercial layer chickens. The keel of poultry is an extension of the abdomen side of the sternum along the sagittal plane and is one of the most important bones. In this study, the keel phenotype of White Leghorns laying hen flocks showed significant individual differences. To clarify its genetic mechanism, we first estimated the heritability of keel bend (KB) in White Leghorn, recorded the production performance of the chicken flock, examined the blood biochemical indexes and bone quality in KB and keel normal (KN) chickens, and performed whole-genome pooled sequencing in KB and KN chickens. We then performed selection elimination analysis to determine the genomic regions that may affect the keel phenotypes. The results show that KB is a medium heritability trait. We found that cage height had a significant effect on the KB (p < 0.01). At 48 weeks, there were significant differences in the number of eggs, the number of normal eggs, and eggshell strength (p < 0.05). The content of parathyroid hormone was lower (p < 0.01) and that of calcitonin was higher (p < 0.01) in KB chickens than in KN chickens. The differences in bone mineral density, bone strength, and bone cortical thickness of the humerus and femur were extremely significant (p < 0.01), with all being lower in KB chickens than in KN chickens. In addition, the bones of KB chickens contained more fat organization. A total of 128 genes were identified in selective sweep regions. We identified 10 important candidate genes: ACP5, WNT1, NFIX, CNN1, CALR, FKBP11, TRAPPC5, MAP2K7, RELA, and ENSGALG00000047166. Among the significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways found, we identifed two bone-related pathways, one involving “osteoclast differentiation” and the other the “MAPK signaling pathway.” These results may help us better understand the molecular mechanism of bone traits in chickens and other birds and provide new insights for the genetic breeding of chickens.

Abstract 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.

Transcriptome analysis has been used to investigate many economically traits in chickens; however, alternative splicing still lacks a systematic method of study that is able to promote proteome diversity, and fine-tune expression dynamics. Hybridization has been widely utilized in chicken breeding due to the resulting heterosis, but the dynamic changes in alternative splicing during this process are significant yet unclear. In this study, we performed a reciprocal crossing experiment involving the White Leghorn and Cornish Game chicken breeds which exhibit major differences in body size and reproductive traits, and conducted RNA sequencing of the brain, muscle, and liver tissues to identify the inheritance patterns. A total of 40 515 and 42 612 events were respectively detected in the brain and muscle tissues, with 39 843 observed in the liver; 2807, 4242, and 4538 events significantly different between two breeds were identified in the brain, muscle, and liver tissues, respectively. The hierarchical cluster of tissues from different tissues from all crosses, based on the alternative splicing profiles, suggests high tissue and strain specificity. Furthermore, a comparison between parental strains and hybrid crosses indicated that over one third of alternative splicing genes showed conserved patterns in all three tissues, while the second prevalent pattern was non-additive, which included both dominant and transgressive patterns; this meant that the dominant pattern plays a more important role than suppression. Our study provides an overview of the inheritance patterns of alternative splicing in layer and broiler chickens, to better understand post-transcriptional regulation during hybridization.

Laying and reproductive performance, egg quality, and disease resistance of hens decrease during the late laying period. Exogenous enzymes promote nutrient digestibility and utilization and improve the intestinal environment. However, the specific regulation of the gut microbiome and metabolome by exogenous enzymes remains unelucidated. This study was conducted to evaluate effects of dietary multi-enzyme supplementation on egg and reproductive performance, egg quality, ileum microbiome, and metabolome of breeders. Here, 224 Hy-Line Brown breeding hens (55 weeks old) were randomly allocated to two groups: dietary controls fed basal diet (DC), and test hens fed 0.2 g/kg corn enzyme diet (CE). Serum levels of total protein, globulin, immunoglobulin Y, and antibodies against the Newcastle disease virus and avian influenza H9 strain were significantly increased (p < 0.05). Egg albumen height, Haugh unit, and fertilization and hatching rates were also significantly increased (p < 0.05) in the CE-fed group. 16S rRNA sequence analysis showed that CE strongly affected both α- and β-diversity of the ileal microbiota. LEfSe analysis revealed that the potentially beneficial genera Lactobacillus, Enterococcus, Faecalicoccus, and Streptococcus were enriched as biomarkers in the CE-fed group. Microbial functional analysis revealed that the functional genes associated with harmful-substance biodegradation was significantly increased in the CE-fed group. Additionally, Spearman correlation analysis indicated that changes in microbial genera were correlated with differential metabolites. In summary, dietary multi-enzyme addition can improve egg quality, humoral immunity, and reproductive performance and regulate the intestinal microbiome and metabolome in breeders. Therefore, multi-enzymes could be used as feed additive to extend breeder service life.

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.

Abstract 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.

Colibacillosis is an acute and chronic avian disease caused by avian pathogenic E. coli (APEC). Previous studies have demonstrated that RIP2 plays a significant role in APEC infection. Moreover, increasing evidence indicates that microRNAs (miRNAs) are involved in host–pathogen interactions and the immune response. However, the role of miRNAs in the host against APEC infection remains unclear. Herein, we attempted to reveal new miRNAs potentially involved in the regulation of the immune and inflammatory response against APEC infection, with a particular focus on those possibly correlated with RIP2 expression, via miRNA-seq, RT-qPCR, Western blotting, dual-luciferase reporter assay, and CCK-8. The results showed that a total of 93 and 148 differentially expressed (DE) miRNAs were identified in the knockdown of RIP2 cells following APEC infection (shRIP2+APEC) vs. knockdown of RIP2 cells (shRIP2) and shRIP2 vs. wild-type cells (WT), respectively. Among those identified DE miRNAs, the biological function of gga-miR-455-5p was investigated. It was found that gga-miR-455-5p regulated by RIP2 was involved in the immune and inflammatory response against APEC infection via targeting of IRF2 to modulate the expression of type I interferons. Additionally, RIP2 could directly regulate the production of the type I interferons. Altogether, these findings highlighted the crucial role of miRNAs, especially gga-miR-455-5p, in host defense against APEC infection.

The regulation of gene expression is a complex process involving organism function and phenotypic diversity, and is caused by cis- and trans- regulation. While prior studies identified the regulatory pattern of the autosome rewiring in hybrids, the role of gene regulation in W sex chromosomes is not clear due to their degradation and sex-limit expression. Here, we developed reciprocal crosses of two chicken breeds, White Leghorn and Cornish Game, which exhibited broad differences in gender-related traits, and assessed the expression of the genes on the W chromosome to disentangle the contribution of cis- and trans-factors to expression divergence. We found that female-specific selection does not have a significant effect on W chromosome gene-expression patterns. For different tissues, there were most parental divergence expression genes in muscle, and also more heterosis compared with two other tissues. Notably, a broader pattern of trans regulation in the W chromosome was observed, which is consistent with autosomes. Taken together, this work describes the regulatory divergence of W-linked genes between two contrasting breeds and indicates sex chromosomes have a unique regulation and expression mechanism.

This study aimed to investigate the effects of dietary yeast culture (YC) supplementation on egg production, egg quality, reproductive performance, immune functions, antioxidant capacity, and intestinal microbial structure of aged hens. A total of 224 Hy-Line Brown layers (54 weeks old) were randomly assigned to two dietary treatments. The control group was fed a basal diet and the YC group was supplemented with YC at 2.0 g/kg of their diet. Each group had seven replicates with 16 hens each. The study was conducted over a period of 8 weeks. Results indicated that YC addition had no significant effect on laying performance. However, it significantly improved egg quality and hatching rate, enhanced ileum crude fat digestibility, increased the serum parameters of lysozyme (LZM) and total antioxidation capacity (T-AOC) (P < 0.05), and reduced serum aspartate aminotransferase (AST) levels (P < 0.05). Using 16S rRNA analysis, we found that addition of YC significantly altered ileum microbial composition. Linear discriminant analysis of effect size (LEfSe) showed significant enrichment of Bacilli and Lactobacilli in the YC group. PICRUSt analysis of the ileal microbiota found that glutathione metabolism, ubiquinone, and other terpenoid-quinone biosynthesis and lipopolysaccharide biosynthesis protein pathways were highly enriched in the YC group compared with the basal diet group. In summary, the addition of YC can improve egg quality, immune functions, antioxidant capacity, reproduction efficiency, and digestive absorption by increasing the abundance of Lactobacilli and Bacilli. Furthermore, it also improves the biosynthesis of lipopolysaccharide proteins, glutathione metabolism, and the synthesis of ubiquinone and other terpenoid-quinone metabolic pathways.

Avian pathogenic E. coli (APEC) can cause localized or systemic infection, resulting in large economic losses per year, and impact health of humans. Previous studies showed that RIP2 (receptor interacting serine/threonine kinase 2) and its signaling pathway played an important role in immune response against APEC infection. In this study, chicken HD11 cells were used as an in vitro model to investigate the function of chicken RIP2 and the transcription factor binding to the RIP2 core promoter region via gene overexpression, RNA interference, RT-qPCR, Western blotting, dual luciferase reporter assay, CHIP-PCR, CCK-8, and flow cytometry assay following APEC stimulation. Results showed that APEC stimulation promoted RIP2 expression and cells apoptosis, and inhibited cells viability. Knockdown of RIP2 significantly improved cell viability and suppressed the apoptosis of APEC-stimulated cells. Transcription factor NFIB (Nuclear factor I B) and GATA1 (globin transcription factor 1) binding site was identified in the core promoter region of RIP2 from −2300 bp to −1839 bp. However, only NFIB was confirmed to be bound to the core promoter of RIP2. Overexpression of NFIB exacerbated cell injuries with significant reduction in cell viability and increased cell apoptosis and inflammatory cytokines levels, whereas opposite results were observed in NFIB inhibition treatment group. Moreover, RIP2 was up-regulated by NFIB overexpression, and RIP2 silence mitigated the effect of NFIB overexpression in cell apoptosis, inflammation, and activation of NFκB signaling pathways. This study demonstrated that NFIB overexpression accelerated APEC-induced apoptosis and inflammation via up-regulation of RIP2 mediated downstream pathways in chicken HD11 cells.

Chicken plumage colour is a complex trait controlled by many genes. Herein, through Rhode Island Red (RIR) and White Leghorn (WL) F1 cross populations, the segregation of plumage color was observed in females, showing white in males, and dark red (DR) and light yellow (LY) in females. The white has been found to be caused by dominant white alleles (I) and the DR phenotype is attributed to a sex-linked recessive silver allele (S∗S). LY is a derived feather colour phenotype and the genetic mechanism of this is unclear. In order to explore the genetic basis for LY, we randomly selected 40 DR and 39 LY chickens for paired-end sequencing. Through the use of association analysis, we found the LY phenotype is caused by a 7.6 kb non-coding deletion near the SOX10 gene. This mutation has been reported to be responsible for dark brown plumage in chicken, and subsequent diagnostic PCR tests showed that the length of the long-range non-coding deletion is 7.6 kb instead of 8.3 kb as previously reported.

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.

Dropping moisture (DM) refers to the water content of feces. High DM in chickens could be disadvantageous to pathogen control and fecal treatment in chicken farms. DM can be affected by environment, nutrition, disease, and genetics. In the present study, significant individual differences were presented in the DM of Rhode Island Red (RIR) chicken population, indicating that genetics could contribute to DM in the chickens. Subsequently, we estimated the genetic parameters of DM and conducted a genome-wide association study (GWAS) to find the potential genomic regions related to DM. The results showed that the heritability of DM ranged from 0.25 to 0.32. Furthermore, 11 significant loci on chromosome 7 were found to be associated with DM levels by the GWAS. The SNP rs15833816 within the COL6A3 gene was the most significant SNP related to DM. Hens carrying the G allele including GA and GG produced higher DM (P < 0.01) levels than those carrying the other genotype AA. Our results showed that DM is a medium-inheritable trait and that COL6A3 could be a potential candidate gene that regulates DM level in chickens.

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.

Molting is natural adaptation to climate change in all birds, including chickens. Forced molting (FM) can rejuvenate and reactivate the reproductive potential of aged hens, but the effect of natural molting (NM) on older chickens is not clear. To explore why FM has a dramatically different effect on chickens compared with NM, the transcriptome analyses of the hypothalamus and ovary in forced molted and natural molted hens at two periods with feathers fallen and regrown were performed. Additionally, each experimental chicken was tested for serological indices. The results of serological indices showed that growth hormone, thyroid stimulating hormone, and thyroxine levels were significantly higher (p < 0.05) in forced molted hens than in natural molted hens, and calcitonin concentrations were lower in the forced molted than in the natural molted hens. Furthermore, the transcriptomic analysis revealed a large number of genes related to disease resistance and anti-aging in the two different FM and NM periods. These regulatory genes and serological indices promote reproductive function during FM. This study systematically revealed the transcriptomic and serological differences between FM and NM, which could broaden our understanding of aging, rejuvenation, egg production, and welfare issues related to FM in chickens.

Molting in birds provides us with an ideal genetic model for understanding aging and rejuvenation since birds present younger characteristics for reproduction and appearance after molting. Forced molting (FM) by fasting in chickens causes aging of their reproductive system and then promotes cell redevelopment by providing water and feed again. To reveal the genetic mechanism of rejuvenation, we detected blood hormone indexes and gene expression levels in the hypothalamus and ovary of hens from five different periods during FM. Three hormones were identified as participating in FM. Furthermore, the variation trends of gene expression levels in the hypothalamus and ovary at five different stages were found to be basically similar using transcriptome analysis. Among them, 45 genes were found to regulate cell aging during fasting stress and 12 genes were found to promote cell development during the recovery period in the hypothalamus. In addition, five hub genes (INO80D, HELZ, AGO4, ROCK2, and RFX7) were identified by WGCNA. FM can restart the reproductive function of aged hens by regulating expression levels of genes associated with aging and development. Our study not only enriches the theoretical basis of FM but also provides insights for the study of antiaging in humans and the conception mechanism in elderly women.

Dropping moisture (DM) refers to the water content in feces. High DM negatively affects poultry production, environment, production costs, and animal health. Heredity, nutrition, environment, and disease may affect DM level. DM has medium inheritability and is related to cage height in henhouses. We examined the relationship among DM level, production performance, and environmental factors at different locations at the same henhouse height and effects of three types of additives. We measured the correlation between environmental factors including temperature, humidity, CO2 concentration, absolute pressure, and DM levels and laying performance of 934 Rhode Island Red hens. DM level was not significantly associated with environmental factors or production performance. We divided 64 persistently high DM hens into control and treatment groups supplied with different additives (probiotics, anisodamine, and antibiotics). DM levels, laying performance, egg quality, and serum biochemical indices were determined. Compared with the control and antibiotics, probiotics significantly reduced DM levels and eggshell strength while improving yolk color but did not significantly affect production performance. The additives reduced the b value of eggshell color; compared with probiotics, anisodamine decreased serum globulin levels. Exogenous active yeast supplementation can significantly reduce DM levels.

Abstract Marek’s disease (MD), caused by Marek’s disease virus (MDV), is a lymphotropic neoplastic disease. Previous miRNAome analysis showed gga-miR-219b was significantly downregulated in MDV-induced lymphoma, and one of its potential target genes, B-cell chronic lymphocytic /lymphoma 11B (BCL11B) was predicted. In this study, we further investigated the function of gga-miR-219b, and the gain/loss of function assay showed gga-miR-219b inhibited cell migration and reduced cell proliferation by promoting apoptosis not by cell cycle arrest. Gga-miR-219b also suppressed expression of two cell invasion-related genes MMP2 and MMP9. The results indicated suppressive effect of gga-miR-219b on MD tumorigenesis. The gene BCL11B was verified as a direct target gene of gga-miR-219b. RNA interference was performed to block BCL11B. As expected, the effects triggered by BCL11B downregulation were in accordance with that triggered by gga-miR-219b overexpression, suggesting that BCL11B was a stimulative regulator of MD transformation. Moreover, both gga-miR-219b and BCL11B influenced the expression of Meq gene, the most important oncogene in MDV. Additionally, gene expression level of anti-apoptotic genes BCL2 and BCL2L1 was downregulated and pro-apoptotic gene TNFSF10 was upregulated in MSB1 cells with gga-miR-219b overexpression or BCL11B knockdown, which suggested gga-miR-219b promoted cell apoptosis via regulating gene expression in the apoptosis pathways.

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.

Marek’s disease (MD) is a highly contagious viral neoplastic disease caused by Marek’s disease virus (MDV), which can lead to huge economic losses in the poultry industry. Recently, microRNAs (miRNAs) have been found in various cancers and tumors. In recent years, 994 mature miRNAs have been identified through deep sequencing in chickens, but only a few miRNAs have been investigated further in terms of their function. Previously, gga-miR-103-3p was found downregulated in MDV-infected samples by using Solexa deep sequencing. In this study, we further verified the expression of gga-miR-103-3p among MDV-infected spleen, MD lymphoma from liver, noninfected spleen, and noninfected liver, by qPCR. The results showed that the expression of gga-miR-103-3p was decreased in MDV-infected tissues, which was consistent with our previous study. Furthermore, two target genes of gga-miR-103-3p, cyclin E1 (CCNE1) and transcription factor Dp-2 (E2F dimerization partner 2) (TFDP2), were predicted and validated by luciferase reporter assay, qPCR, and western blot analysis. The results suggested that CCNE1 and TFDP2 are direct targets of gga-miR-103-3p in chickens. Subsequent cell proliferation and migration assay showed that gga-miR-103-3p suppressed MDCC-MSB1 migration, but did not obviously modulate MDCC-MSB1 cell proliferation. In conclusion, gga-miR-103-3p targets the CCNE1 and TFDP2 genes, and suppresses cell migration, which indicates that it might play an important role in MD tumor transformation.

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.

Egg number (EN), egg laying rate (LR) and age at first egg (AFE) are important production traits related to egg production in poultry industry. To better understand the knowledge of genetic architecture of dynamic EN during the whole laying cycle and provide the precise positions of associated variants for EN, LR and AFE, laying records from 21 to 72 weeks of age were collected individually for 1,534 F2 hens produced by reciprocal crosses between White Leghorn and Dongxiang Blue-shelled chicken, and their genotypes were assayed by chicken 600 K Affymetrix high density genotyping arrays. Subsequently, pedigree and SNP-based genetic parameters were estimated and a genome-wide association study (GWAS) was conducted on EN, LR and AFE. The heritability estimates were similar between pedigree and SNP-based estimates varying from 0.17 to 0.36. In the GWA analysis, we identified nine genome-wide significant loci associated with EN of the laying periods from 21 to 26 weeks, 27 to 36 weeks and 37 to 72 weeks. Analysis of GTF2A1 and CLSPN suggested that they influenced the function of ovary and uterus, and may be considered as relevant candidates. The identified SNP rs314448799 for accumulative EN from 21 to 40 weeks on chromosome 5 created phenotypic differences of 6.86 eggs between two homozygous genotypes, which could be potentially applied to the molecular breeding for EN selection. Moreover, our finding showed that LR was a moderate polygenic trait. The suggestive significant region on chromosome 16 for AFE suggested the relationship between sex maturity and immune in the current population. The present study comprehensively evaluates the role of genetic variants in the development of egg laying. The findings will be helpful to investigation of causative genes function and future marker-assisted selection and genomic selection in chickens.

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.