The comb, as a secondary sexual character, is an important trait in chicken. Indicators of comb length (CL), comb height (CH), and comb weight (CW) are often selected in production. DNA-based marker-assisted selection could help chicken breeders to accelerate genetic improvement for comb or related economic characters by early selection. Although a number of quantitative trait loci (QTL) and candidate genes have been identified with advances in molecular genetics, candidate genes underlying comb traits are limited. The aim of the study was to use genome-wide association (GWA) studies by 600 K Affymetrix chicken SNP arrays to detect genes that are related to comb, using an F2 resource population. For all comb characters, comb exhibited high SNP-based heritability estimates (0.61-0.69). Chromosome 1 explained 20.80% genetic variance, while chromosome 4 explained 6.89%. Independent univariate genome-wide screens for each character identified 127, 197, and 268 novel significant SNPs with CL, CH, and CW, respectively. Three candidate genes, VPS36, AR, and WNT11B, were determined to have a plausible function in all comb characters. These genes are important to the initiation of follicle development, gonadal growth, and dermal development, respectively. The current study provides the first GWA analysis for comb traits. Identification of the genetic basis as well as promising candidate genes will help us understand the underlying genetic architecture of comb development and has practical significance in breeding programs for the selection of comb as an index for sexual maturity or reproduction.

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 weight (EW) is an economically-important trait and displays a consecutive increase with the hen's age. Because extremely large eggs cause a range of problems in the poultry industry, we performed a genome-wide association study (GWAS) in order to identify genomic variations that are associated with EW. We utilized the Affymetrix 600 K high density SNP array in a population of 1,078 hens at seven time points from day at first egg to 80 weeks age (EW80). Results reveal that a 90 Kb genomic region (169.42 Mb ~ 169.51 Mb) in GGA1 is significantly associated with EW36 and is also potentially associated with egg weight at 28, 56, and 66 week of age. The leading SNP could account for 3.66% of the phenotypic variation, while two promising genes (DLEU7 and MIR15A) can be mapped to this narrow significant region and may affect EW in a pleiotropic manner. In addition, one gene (CECR2 on GGA1) and two genes (MEIS1 and SPRED2 on GGA3), which involved in the processes of embryogenesis and organogenesis, were also considered to be candidates related to first egg weight (FEW) and EW56, respectively. Findings in our study could provide worthy theoretical basis to generate eggs of ideal size based on marker assisted breeding selection.

Blood biochemical indicators play a crucial role in assessing an individual's overall health status and metabolic function. In this study, we measured five blood biochemical indicators, including total cholesterol (CHOL), low-density lipoprotein cholesterol (LDL-CH), triglycerides (TG), high-density lipoprotein cholesterol (HDL-CH), and blood glucose (BG), as well as 19 growth traits of 206 male chickens. By integrating host whole-genome information and 16S rRNA sequencing of the duodenum, jejunum, ileum, cecum, and feces microbiota, we assessed the contributions of host genetics and gut microbiota to blood biochemical indicators and their interrelationships. Our results demonstrated significant negative phenotypic and genetic correlations (r =  - 0.20 ~  - 0.67) between CHOL and LDL-CH with growth traits such as body weight, abdominal fat content, muscle content, and shin circumference. The results of heritability and microbiability indicated that blood biochemical indicators were jointly regulated by host genetics and gut microbiota. Notably, the heritability of HDL-CH was estimated to be 0.24, while the jejunal microbiability for BG and TG reached 0.45 and 0.23. Furthermore, by conducting genome-wide association study (GWAS) with the single-nucleotide polymorphism (SNPs), insertion/deletion (indels), and structural variation (SV), we identified RAP2C, member of the RAS oncogene family (RAP2C), dedicator of cytokinesis 11 (DOCK11), neurotensin (NTS) and BOP1 ribosomal biogenesis factor (BOP1) as regulators of HDL-CH, and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5), dihydrodiol dehydrogenase (DHDH), and potassium voltage-gated channel interacting protein 1 (KCNIP1) as candidate genes of BG. Moreover, our findings suggest that cecal RF39 and Clostridia_UCG_014 may be linked to the regulation of CHOL, and jejunal Streptococcaceae may be involved in the regulation of TG. Additionally, microbial GWAS results indicated that the presence of gut microbiota was under host genetic regulation. Our findings provide valuable insights into the complex interaction between host genetics and microbiota in shaping the blood biochemical profile of chickens. KEY POINTS: • Multiple candidate genes were identified for the regulation of CHOL, HDL-CH, and BG. • RF39, Clostridia_UCG_014, and Streptococcaceae were implicated in CHOL and TG modulation. • The composition of gut microbiota is influenced by host genetics.

Enterotypes are used to describe clusters of specific gut microbial community structures, but few reports exist on the identification of enterotypes in poultry. In addition, there is incomplete understanding on the role of the foregut microbiota in the digestion and absorption of nutrients in poultry. Thus, this study aimed to identify the duodenal enterotypes by examining microbial communities from 206 broilers using 16S rRNA high-throughput sequencing and explore the effects of enterotypes on phenotypic performance and nutrient metabolism with metabolomics. The duodenal microbial communities of the broiler population were partitioned into 3 enterotypes (ET1, ET2, and ET3), and significant differences were observed in α-diversity among the enterotypes (P < 0.01). At the genus level, the ET1 group was over-represented by Bacteroides (9.8%) and Escherichia-Shigella (8.9%), the ET2 group was over-represented by Ochrobactrum (19.4%) and Rhodococcus (14.7%), and the ET3 group was over-represented by Bacillus (23.4%) and Akkermansia (16.2%). The relative abundance of the dominant taxa of each enterotype was significantly higher than that in the other 2 enterotypes (P < 0.01). The results showed that Ochrobactrum and Rhodococcus were positively correlated with cellobiose, alpha-D-glucose, D-mannose, and D-allose (r = 0.429, 0.435, 0.482, and 0.562, respectively; all P < 0.05). Rhodococcus was also positively correlated with tridecanoic acid and glycerol 1-myristate (r = 0.655 and 0.489, respectively; all P < 0.01). In terms of phenotype, the triglyceride level in the ET2 group was significantly higher than that in the ET1 group (P < 0.05), and the subcutaneous fat thickness and abdominal fat weight in the ET2 group were the highest (P > 0.05). Taken together, these results confirmed the presence of enterotypes in broilers and found that the dominant microbes in broilers of the ET2 group might play a major role in the degradation and utilization of plant polysaccharides, which may have an impact on the serum triglyceride level and fat deposition in broilers. These findings lay a foundation for further studies on the gut microbial interactions with the metabolism in broilers and the regulation of the gut microbiota to promote growth and well-being in broilers.

As a major economic trait in chickens, egg weight (EW) receives widespread interests in breeding, production and consumption. However, limited information is available for underlying genetic architecture of longitudinal trend in EW. Herein, we measured EWs at nine time points from onset of laying to 60 week of age, and conducted comprehensive genome-wide association studies (GWAS) in 1,534 F2 hens derived from reciprocal crosses between White Leghorn and Dongxiang chickens.

With the development of molecular genetics and high-throughput sequencing technology, genotyping arrays consisting of large numbers of SNP have raised great interest in animal and plant research. However, the application of commercial chicken 600K SNP arrays has varied in different populations of egg-type chickens. Moreover, their genotyping cost is too high for large-scale population applications. Herein, we independently developed a custom Illumina 50K BeadChip, named PhenoixChip-I, for egg-type chickens based on SNP from 479 sequenced individuals in 7 lines. We filtered and selected SNP with stringent criteria, such as high polymorphism, genome coverage, design score, and priorities. Finally, a total of 43,681 effective SNP successfully genotyped were included on our custom array. Approximately 14K SNP were previously reported to be associated with important economic traits in egg-type chickens. Subsequently, we verified the applicability and efficiency of the PhenoixChip-I SNP array from many aspects, including evaluating its use scientific research (population structure analysis and genome-wide association study) and the poultry breeding industry (genomic selection). The findings in our study will play a crucial role in accelerating the genetic improvement of egg-type chickens.

Molecular genetic tools provide a method for improving the breeding selection of chickens (Gallus gallus). Although some studies have identified genes affecting egg quality, little is known about the genes responsible for oviduct development. To address this issue, here we used a genome-wide association (GWA) study to detect genes or genomic regions that are related to oviduct development in a chicken F2 resource population by employing high-density 600 K single-nucleotide polymorphism (SNP) arrays. For oviduct length and weight, which exhibited moderate heritability estimates of 0.35 and 0.39, respectively, chromosome 1 (GGA1) explained 9.45% of the genetic variance, while GGA4 to GGA8 and GGA11 explained over 1% of the variance. Independent univariate genome-wide screens for oviduct length and weight detected 69 significant SNPs on GGA1 and 49 suggestive SNPs on GGA1, GGA4, and GGA8. One hundred and fourteen suggestive SNPs were associated with oviduct length, while 73 SNPs were associated with oviduct weight. The significant genomic regions affecting oviduct weight ranged from 167.79-174.29 Mb on GGA1, 73.16-75.70 Mb on GGA4, and 4.88-4.92 Mb on GGA8. The genes CKAP2, CCKAR, NCAPG, IGFBP3, and GORAB were shown to have potential roles in oviduct development. These genes are involved in cell survival, appetite, and growth control. Our results represent the first GWA analysis of genes controlling oviduct weight and length. The identification of genomic loci and potential candidate genes affecting oviduct development greatly increase our understanding of the genetic basis underlying oviduct development, which could have an impact on the selection of egg quality.

Female sperm storage (FSS), the maintenance of sperm inside the female reproductive tract for an extended period of time, is pervasive among organisms with internal fertilization. Because FSS enables asynchronous mating and fertilization, it could be extremely important to reproduction. However, the physiological mechanisms underlying prolonged preservation and maintenance are poorly understood. Here, we used chicken, a typical oviparous animal, to determine the mechanisms ensuring sperm functionality in sperm storage tubules (SSTs).

Heterosis, a phenomenon characterized by the superior performance of hybrid individuals relative to their parents, has been widely utilized in livestock and crop breeding, while the underlying genetic basis remains elusive in chickens. Here, we performed a reciprocal crossing experiment with broiler and layer chickens and conducted RNA sequencing on liver tissues for reciprocal crosses and their parental lines to identify inheritance patterns of gene expression. Our results showed that heterosis of the abdominal fat percentage was 69.28%-154.71% in reciprocal crosses. Over-dominant genes of reciprocal crosses were significantly enriched in three biological pathways, namely, butanoate metabolism, the synthesis and degradation of ketone bodies, and valine, leucine, and isoleucine degradation. Among these shared over-dominant genes, we found that a lipid-related gene, HMGCL, was enriched in these pathways. Furthermore, we validated this gene as over-dominant using qRT-PCR. Although no shared significant pathway was detected in the high-parent dominant genes of reciprocal crosses, high-parent dominant gene expression was the major gene inheritance pattern in reciprocal crosses and we could not exclude the effect of high-parent dominant genes. These findings suggest that non-additive genes play important roles in the heterosis of important traits in chickens and have important implications regarding our understanding of heterosis.

Feed contributes most to livestock production costs. Improving feed efficiency is crucial to increase profitability and sustainability for animal production. Host genetics and the gut microbiota can both influence the host phenotype. However, the association between the gut microbiota and host genetics and their joint contribution to feed efficiency in chickens is largely unclear.

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

Embryonic diapause (dormancy) is a state of temporary arrest of embryonic development that is triggered by unfavorable conditions and serves as an evolutionary strategy to ensure reproductive survival. Unlike maternally-controlled embryonic diapause in mammals, chicken embryonic diapause is critically dependent on the environmental temperature. However, the molecular control of diapause in avian species remains largely uncharacterized. In this study, we evaluated the dynamic transcriptomic and phosphoproteomic profiles of chicken embryos in pre-diapause, diapause, and reactivated states.

Egg production is the most economically-important trait in layers as it directly influences benefits of the poultry industry. To better understand the genetic architecture of egg production, we measured traits including age at first egg (AFE), weekly egg number (EN) from onset of laying eggs to 80 weeks which was divided into five stage (EN1: from onset of laying eggs to 23 weeks, EN2: from 23 to 37 weeks, EN3: from 37 to 50 weeks, EN4: from 50 to 61 weeks, EN5: from 61 to 80 weeks) based on egg production curve and total egg number across the whole laying period (Total-EN). Then we performed genome-wide association studies (GWAS) in 1078 Rhode Island Red hens using a linear mixed model.

Eggshell is a bio-ceramic material comprising columnar calcite (CaCO3) crystals and organic proteinaceous matrix. The size, shape and orientation of the CaCO3 crystals influence the microstructural properties of chicken eggshells. However, the genetic architecture underlying eggshell crystal polymorphism remains to be elucidated.

Improving feed efficiency is one of the main goals of chicken breeding and production. The function of the digestive system, where feed is digested and nutrients are absorbed, is closely related to feed efficiency. However, the association between feed efficiency and the development of different digestive organs in chickens remains unclear. Here, we investigated the individual feed efficiency of 207 broilers during the fast-growing period with an electronic feeder and examined the characteristics of 8 organs of their digestive system (the liver, bile, proventriculus, gizzard, duodenum, jejunum, ileum, and cecum) at market age. Both the feed conversion ratio (FCR) and residual feed intake (RFI) were significantly negatively correlated with the gizzard weight (GW) and significantly positively correlated with the relative weight of the liver (RLW). Additionally, we found an obvious negative relationship between the FCR and cecal length (CL). A two-tailed t test further confirmed these correlation analysis results. Specifically, compared to birds with the lowest feed efficiencies, the GW of broilers with the highest feed efficiencies (the lowest FCR or RFI) was 22.74% and 17.97% higher, respectively. The RLW of chickens with the highest feed efficiencies was 10.82 to 13.73% less than that of chickens with the lowest feed efficiencies. In addition, we found that increased CL (5.42-12.09%) was significantly associated with better feed efficiency. Thus, our study showed that the feed efficiency of broilers was related to the development of the gizzard, liver, and cecum. These findings provide new insight into the genetic and physiological regulation of feed efficiency in broilers.

Hepatic steatosis is a prevalent manifestation of fatty liver, that has detrimental effect on the health and productivity of laying hens, resulting in economic losses to the poultry industry. Here, we aimed to systematically investigate the genetic regulatory mechanisms of hepatic steatosis in laying hens.

Feed contributes to over 60 % of the total production costs in the poultry industry. Increasing feed costs prompt geneticists to include feed intake and efficiency as selection goals in breeding programs. In the present study, we used an F2 chicken population in a genome-wide association study (GWAS) to detect potential genetic variants and candidate genes associated with daily feed intake (FI) and feed efficiency, including residual feed intake (RFI) and feed conversion ratio (FCR).

Breeding of genetically resistant chickens to Marek's disease (MD) is a vital strategy to poultry health. To find the markers underlying the genetic resistance to MD, copy number variation (CNV) was examined in inbred MD-resistant and -susceptible chicken lines. A total of 45 CNVs were found in four lines of chickens, and 28 were potentially involved in immune response and cell proliferation, etc. Importantly, two CNVs related with MD resistance were transmitted to descendent recombinant congenic lines that differ in susceptibility to MD. Our findings may lead to better strategies for genetic improvement of disease resistance in poultry.

Skeletal problems in layer chickens are gaining attention due to animal welfare and economic losses in the egg industry. The genetic improvement of bone traits has been proposed as a potential solution to these issues; however, genetic architecture is not well understood. We conducted a genome-wide association study (GWAS) on bone quality using a sample of 1534 hens genotyped with a 600 K Chicken Genotyping Array. Using a linear mixed model approach, a novel locus close to GSG1L, associated with femur bone mineral density (BMD), was uncovered in this study. In addition, nine SNPs in genes were associated with bone quality. Three of these genes, RANKL, ADAMTS and SOST, were known to be associated with osteoporosis in humans, which makes them good candidate genes for osteoporosis in chickens. Genomic partitioning analysis supports the fact that common variants contribute to the variations of bone quality. We have identified several strong candidate genes and genomic regions associated with bone traits measured in end-of-lay cage layers, which accounted for 1.3-7.7% of the phenotypic variance. These SNPs could provide the relevant information to help elucidate which genes affect bone quality in chicken.