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ABSTRACT: Seminal fluid, once believed to be sterile, is now recognized as constituting a complex and dynamic environment inhabited by a diverse community of micro-organisms. However, research on the seminal microbiota in chickens is limited, and microbiota variations among different chicken breeds remain largely unexplored. In this study, we collected semen samples from Beijing You Chicken (BYC) and Tibetan Chicken (TC) and explored the characteristics of the microbiota using 16S rRNA gene sequencing. Additionally, we collected cloacal samples from the TC to control for environmental contamination. The results revealed that the microbial communities in the semen were significantly different from those in the cloaca. Firmicutes and Actinobacteriota were the predominant phyla in BYC and TC semen, respectively, with Lactobacillus and Phyllobacterium being the dominant genera in each group. Additionally, the seminal microbiota of BYC exhibited greater richness and evenness than that of TC. Principal coordinate analysis (PCoA) indicated significant intergroup differences between the seminal microbiotas of BYC and TC. Subsequently, by combining linear discriminant analysis effect size and random forest analyses, we identified Lactobacillus as the predominant microorganism in BYC semen, whereas Phyllobacterium dominated in TC semen. Furthermore, co-occurrence network analysis revealed a more intricate network in the BYC group than in the TC group. Additionally, unique microbial functional characteristics were observed in each breed, with TC exhibiting metabolic features potentially associated with their ability to adapt to high-altitude environments. The results of this study emphasized the unique microbiota present in chicken semen, which may be influenced by genetics and evolutionary history. Significant variations were observed between low-altitude and high-altitude breeds, highlighting the breed-specific implications of the seminal microbiota for reproduction and high-altitude adaptation.

ABSTRACT: Long-term intensive genetic selection has led to significant differences between broiler and layer chickens, which are evident during the embryonic period. Despite this, there is a paucity of research on the genetic regulation of the initial formation of muscle fiber morphology in chick embryos. Embryonic d 17 (E17) is the key time point for myoblast fusion completion and muscle fiber morphology formation in chickens. This study aimed to explore the genetic regulatory mechanisms underlying the early muscle fiber morphology establishment in broiler chickens of Cornish (CC) and White Plymouth Rock (RR) and layer chickens of White Leghorn (WW) at E17 using the transcriptomic and chromatin accessibility sequencing of pectoral major muscles. The results showed that broiler chickens exhibited significant higher embryo weight and pectoral major muscle weight at E17 compared to layer chickens (P = 0.000). A total of 1,278, 1,248, and 892 differentially expressed genes (DEGs) of RNA-seq data were identified between CC vs. WW, RR vs. WW, and CC vs. RR, separately. All DEGs were combined for cluster analysis and they were divided into 6 clusters, including cluster 1 with higher expression in broilers and cluster 6 with higher expression in layers. DEGs in cluster 1 were enriched in terms related to macrophage activation (P = 0.002) and defense response to bacteria (P = 0.002), while DEGs in cluster 6 showed enrichment in protein-DNA complex (P = 0.003) and monooxygenase activity (P = 0.000). ATAC-seq data analysis identified a total of 38,603 peaks, with 13,051 peaks for CC, 18,780 peaks for RR, and 6,772 peaks for WW. Integrative analysis of transcriptomic and chromatin accessibility data revealed GOLM1, ISLR2, and TOPAZ1 were commonly upregulated genes in CC and RR. Furthermore, screening of all upregulated DEGs in cluster 1 from CC and RR identified GOLM1, ISLR2, and HNMT genes associated with neuroimmune functions and MYOM3 linked to muscle morphology development, showing significantly elevated expression in broiler chickens compared to layer chickens. These findings suggest active neural system connectivity during the initial formation of muscle fiber morphology in embryonic period, highlighting the early interaction between muscle fiber formation morphology and the nervous system. This study provides novel insights into late chick embryo development and lays a deeper foundation for further research.

ABSTRACT: The industry of egg-type chicken has shown a trend of extending the rearing period, with the goal of breeding chicken breeds capable of producing 500 qualified eggs by 700 d of age. However, the rapid decline in eggshell quality during the late laying period is one of the major challenges. In this study, a total of 3,261 Rhode Island Red chickens were used to measure eggshell quality traits including eggshell strength (ESS), eggshell thickness (EST), eggshell color (ESC) and eggshell gloss (ESG) at seven age points ranging from 36 to 90 wk of age. Phenotypic variations increased with the aging process, especially during the late laying period (> 55 wk), and the heritability during this period decreased by 22.7 to 81.4% compared to the initial and peak laying periods. Then we performed genome-wide association study (GWAS) to identify the genomic variants that associated with eggshell quality, with a custom Illumina 50K BeadChip, named PhenoixChip-I. The results indicated that 2 genomic regions on GGA1(23.24-25.15Mb; 175.95-176.05 Mb) were significantly (P < 4.48E-06) or suggestively (P < 8.97E-05) associated with ESS, which can explain 9.59% and 0.48% of the phenotypic variations of ESS46 and ESS36, respectively. Three genes, FRY, PCNX2, and ENSGALG00000052468, were considered to be the candidate genes for ESS. For other traits, the genome-wide suggestive SNPs were identified at each age point, exhibiting a certain trend with aging process. Additionally, SNP enrichment analysis and functional annotation of cross-tissue regulatory elements to ESS36 revealed a high concentration of enhancer elements specific to shell gland and kidney tissues. This study, deepened our knowledge of eggshells and laying a valued scientific foundation for chicken molecular breeding.

Abstract Background Sex determination and differentiation are complex and delicate processes. In female chickens, the process of sex differentiation is sensitive and prone to be affected by the administration of aromatase inhibitors, which result in chicken sex reversal and infertility. However, the molecular mechanisms underlying sex differentiation and infertility in chicken sex reversal remain unclear. Therefore, we established a sex-reversed chicken flock by injecting an aromatase inhibitor, fadrozole, and constructed relatively high-resolution profiles of the gene expression and chromatin accessibility of embryonic gonads. Results We revealed that fadrozole affected the transcriptional activities of several genes, such as DMRT1, SOX9, FOXL2, and CYP19A1, related to sex determination and differentiation, and the expression of a set of gonadal development-related genes, such as FGFR3 and TOX3, by regulating nearby open chromatin regions in sex-reversed chicken embryos. After sexual maturity, the sex-reversed chickens were confirmed to be infertile, and the possible causes of this infertility were further investigated. We found that the structure of the gonads and sperm were greatly deformed, and we identified several promising genes related to spermatogenesis and infertility, such as SPEF2, DNAI1, and TACR3, through RNA-seq. Conclusions This study provides clear insights into the exploration of potential molecular basis underlying sex differentiation and infertility in sex-reversed chickens and lays a foundation for further research into the sex development of birds.

ABSTRACT: Marek's disease (MD) is a lymphoproliferative neoplastic disease caused by Marek's disease virus (MDV). Previous studies have showed that DNA methylation was involved in MD development, but systematic studies are still lacking. Herein, we performed whole genome bisulfite sequencing (WGBS) and RNA-seq in MDV-infected tumorous spleens (IN), noninfected spleens (NoIN), and survivor (SUR) spleens of chickens to identify the genes playing important roles in MD tumor transformation. We generated the first genome-wide DNA methylation profile of MDV-infected, noninfected, and survivor chickens. Combined the WGBS and RNA-Seq, we found that the expression of 25% differential expression genes (DEGs) were significantly correlated with methylation of CpG sites in their gene bodies or promoters. Further, we focused on the DEGs with differentially methylated regions (DMRs) on genes’ body and promoter, and it showed the expression of 60% DEGs were significantly correlated with methylation of CpG sites in DMRs. Finally, we identified 8 genes, including CD4, CTLA4, DTL, HMGB1, LGMN, NUP210, RAD52, and ZAP70, and their expression was negatively correlated with methylation of DMRs in their promoters in both IN vs. NoIN and IN vs. SUR. These 8 genes showed specifically high expression in IN groups and clustered in module turquoise analyzed by WGCNA. Out of 8 genes, CD4 and HMGB1 were drop in QTLs associated with MD resistance. Thus, we overexpressed the 2 genes to simulate their high expression in the IN group and found they significantly promoted MDCC-MSB-1 cell proliferation, which revealed they might play promoting roles in MD tumorigenesis in IN due to their high expression induced by hypomethylation.

Abstract Background Multi-omics data can provide a stereoscopic view to explore potential causal variations and genes, as well as underlying genetic mechanisms of complex traits. However, for many non-mammalian species, including chickens, these resources are poorly integrated and reused, greatly limiting genetic research and breeding processes of the species. Results Here, we constructed Galbase, an easily accessible repository that integrates public chicken multi-omics data from 928 re-sequenced genomes, 429 transcriptomes, 379 epigenomes, 15,275 QTL entries, and 7,526 associations. A total of 21.67 million SNPs, 2.71 million InDels, and 488,583 cis-regulatory elements were included. Galbase allows users to retrieve genomic variations in geographical maps, gene expression profiling in heatmaps, and epigenomic signals in peak patterns. It also provides modules for batch annotation of genes, regions, and loci based on multi-layered omics data. Additionally, a series of convenient tools, including the UCSC Genome Browser, WashU Epigenome Browser, BLAT, BLAST, and LiftOver, were also integrated to facilitate search, visualization, and analysis of sequence features. Conclusion Galbase grants new opportunities to research communities to undertake in-depth functional genomic studies on chicken. All features of Galbase make it a useful resource to identify genetic variations responsible for chicken complex traits. Galbase is publicly available at http://animal.nwsuaf.edu.cn/ChickenVar .

Abstract Background As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. Results The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. Conclusions This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

Abstract Background 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. Results The integral intensity of the nine major diffraction peaks, total integral intensity and degree of orientation of the crystals were measured followed by a genome-wide association study in 839 F2 hens. The results showed that the total integral intensity was positively correlated with the eggshell strength, eggshell thickness, eggshell weight, mammillary layer thickness and effective layer thickness. The SNP-based heritabilities of total integral intensity and degree of orientation were 0.23 and 0.06, respectively. The 621 SNPs located in the range from 55.6 to 69.1 Mb in GGA1 were significantly associated with TA. PLCZ1, ABCC9, ITPR2, KCNJ8, CACNA1C and IAPP, which are involved in the biological process of regulating cytosolic calcium ion concentration, can be suggested as key genes regulating the total integral intensity. Conclusions The findings greatly advance the understanding of the genetic basis underlying the crystal ultrastructure of eggshell quality and thus will have practical significance in breeding programs for improving eggshell quality.

Sex determination and differentiation are processes by which a bipotential gonad adopts either a testicular or ovarian cell fate, and secondary sexual characteristics adopt either male or female developmental patterns. In birds, although genetic factors control the sex determination program, sex differentiation is sensitive to hormones, which can induce sex reversal when disturbed. Although these sex-reversed birds can form phenotypes opposite to their genotypes, none can experience complete sex reversal or produce offspring under natural conditions. Promising evidence indicates that the incomplete sex reversal is associated with cell autonomous sex identity (CASI) of avian cells, which is controlled by genetic factors. However, studies cannot clearly describe the regulatory mechanism of avian CASI and sex development at present, and these factors require further exploration. In spite of this, the abundant findings of avian sex research have provided theoretical bases for the progress of gender control technologies, which are being improved through interdisciplinary co-operation and will ultimately be employed in poultry production. In this review, we provide an overview of avian sex determination and differentiation and comprehensively summarize the research progress on sex reversal in birds, especially chickens. Importantly, we describe key issues faced by applying gender control systems in poultry production and chronologically summarize the development of avian sex control methods. In conclusion, this review provides unique perspectives for avian sex studies and helps scientists develop more advanced systems for sex regulation in birds.

Abstract Background Poultry is the major reservoir of Campylobacter that contributes to human campylobacteriosis and threatens food safety. Litter contact has been linked to Campylobacter colonization, but the gut microecological impact underlying this link remains not fully clear. Here, we sought to investigate the impact of the gut microecology on the presence of Campylobacter by examining the microbiota in the duodenum, jejunum, ileum, ceca, and feces from chickens raised on commercial litter and in individual cages at 0–57 days of age. Results Through litter contact, the presence of Campylobacter was found to benefit from microecological competition among Lactobacillus, Helicobacter, and genera that are halotolerant and aerobic or facultatively anaerobic in the upper intestine, such as Corynebacterium and Brachybacterium. The presence was also promoted by the increased abundance in obligate anaerobic fermentation microbes, especially members of the orders Clostridiales and Bacteroidales. The longitudinal analysis supported the vertical or pseudo-vertical transmission but suggested that colonization might occur immensely at 7–28 days of age. We observed a host genetic effect on the gut microecology, which might lead to increased heterogeneity of the microecological impact on Campylobacter colonization. Conclusions The findings advance the understanding of the gut microecological impact on Campylobacter presence in the chicken gut under conditions of litter contact and suggest that manipulations of the gut microecology, as well as the microbes identified in the Campylobacter association networks, might be important for the development of intervention strategies.

Abstract Background 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. Results Here, we examined microbial data from the duodenum, jejunum, ileum, cecum, and feces in 206 chickens and their host genotypes and confirmed that the microbial phenotypes and co-occurrence networks exhibited dramatic spatial heterogeneity along the digestive tract. The correlations between host genetic kinship and gut microbial similarities within different sampling sites were weak, with coefficients ranging from − 0.07 to 0.08. However, microbial genome-wide analysis revealed that genetic markers near or inside the genes MTHFD1L and LARGE1 were associated with the abundances of cecal Megasphaera and Parabacteroides, respectively. The effect of host genetics on residual feed intake (RFI) was 39%. We further identified three independent genetic variations that were related to feed efficiency and had a modest effect on the gut microbiota. The contributions of the gut microbiota from the different parts of the intestinal tract on RFI were distinct. The cecal microbiota accounted for 28% of the RFI variance, a value higher than that explained by the duodenal, jejunal, ileal, and fecal microbiota. Additionally, six bacteria exhibited significant associations with RFI. Specifically, lower abundances of duodenal Akkermansia muciniphila and cecal Parabacteroides and higher abundances of cecal Lactobacillus, Corynebacterium, Coprobacillus, and Slackia were related to better feed efficiency. Conclusions Our findings solidified the notion that both host genetics and the gut microbiota, especially the cecal microbiota, can drive the variation in feed efficiency. Although host genetics has a limited effect on the entire microbial community, a small fraction of gut microorganisms tends to interact with host genes, jointly contributing to feed efficiency. Therefore, the gut microbiota and host genetic variations can be simultaneously targeted by favoring more-efficient taxa and selective breeding to improve feed efficiency in chickens. Video abstract

Abstract Background Heterosis is an important biological phenomenon that has been extensively utilized in agricultural breeding. However, negative heterosis is also pervasively observed in nature, which can cause unfavorable impacts on production performance. Compared with systematic studies of positive heterosis, the phenomenon of negative heterosis has been largely ignored in genetic studies and breeding programs, and the genetic mechanism of this phenomenon has not been thoroughly elucidated to date. Here, we used chickens, the most common agricultural animals worldwide, to determine the genetic and molecular mechanisms of negative heterosis. Results We performed reciprocal crossing experiments with two distinct chicken lines and found that the body weight presented widely negative heterosis in the early growth of chickens. Negative heterosis of carcass traits was more common than positive heterosis, especially breast muscle mass, which was over − 40% in reciprocal progenies. Genome-wide gene expression pattern analyses of breast muscle tissues revealed that nonadditivity, including dominance and overdominace, was the major gene inheritance pattern. Nonadditive genes, including a substantial number of genes encoding ATPase and NADH dehydrogenase, accounted for more than 68% of differentially expressed genes in reciprocal crosses (4257 of 5587 and 3617 of 5243, respectively). Moreover, nonadditive genes were significantly associated with the biological process of oxidative phosphorylation, which is the major metabolic pathway for energy release and animal growth and development. The detection of ATP content and ATPase activity for purebred and crossbred progenies further confirmed that chickens with lower muscle yield had lower ATP concentrations but higher hydrolysis activity, which supported the important role of oxidative phosphorylation in negative heterosis for growth traits in chickens. Conclusions These findings revealed that nonadditive genes and their related oxidative phosphorylation were the major genetic and molecular factors in the negative heterosis of growth in chickens, which would be beneficial to future breeding strategies.

In birds, male gonads form on both sides whereas most females develop asymmetric gonads. Multiple early lines of evidence suggested that the right gonad fails to develop into a functional ovary, mainly due to differential expression of PITX2 in the gonadal epithelium. Despite some advances in recent years, the molecular mechanisms underlying asymmetric gonadal development remain unclear. Here, using bulk analysis of whole gonads, we established a relatively detailed profile of four representative stages of chicken gonadal development at the transcriptional and chromatin levels. We revealed that many candidate genes were significantly enriched in morphogenesis, meiosis and subcellular structure formation, which may be responsible for asymmetric gonadal development. Further chromatin accessibility analysis suggested that the transcriptional activities of the candidate genes might be regulated by nearby open chromatin regions, which may act as transcription factor (TF) binding sites and potential cis-regulatory elements. We found that LHX9 was a promising TF that bound to the left-biased peaks of many cell cycle-related genes. In summary, this study provides distinctive insights into the potential molecular basis underlying the asymmetric development of chicken gonads.

Abstract Background Marek’s disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD. Results In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study. Conclusions Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

ABSTRACT: 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.

Abstract Background 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. Results Estimates of pedigree and SNP-based genetic parameter showed that AFE and EN1 exhibited high heritability (0.51 ± 0.09, 0.53 ± 0.08), while the h2 for EN in other stages varied from low (0.07 ± 0.04) to moderate (0.24 ± 0.07) magnitude. Subsequently, seven univariate GWAS for AFE and ENs were carried out independently, from which a total of 161 candidate SNPs located on GGA1, GGA2, GGA5, GGA6, GGA9 and GGA24 were identified. Thirteen SNP located on GGA6 were associated with AFE and an interesting gene PRLHR that may affect AFE through regulating oxytocin secretion in chickens. Sixteen genome-wide significant SNPs associated with EN3 were in a strong linkage disequilibrium (LD) region spanning from 117.87 Mb to 118.36 Mb on GGA1 and the most significant SNP (rs315777735) accounted for 3.57% of phenotypic variance. Genes POLA1, PDK3, PRDX4 and APOO identified by annotating sixteen genome-wide significant SNPs can be considered as candidates associated with EN3. Unfortunately, our study did not find any candidate gene for the total egg number. Conclusions Findings in our study could provide promising genes and SNP markers to improve egg production performance based on marker-assisted breeding selection, while further functional validation is still needed in other populations.

The basic units of skeletal muscle in all vertebrates are multinucleate myofibers, which are formed from the fusion of mononuclear myoblasts during the embryonic period. In order to understand the regulation of embryonic muscle development, we selected four chicken breeds, namely, Cornish (CN), White Plymouth Rock (WPR), White Leghorn (WL), and Beijing-You Chicken (BYC), for evaluation of their temporal expression patterns of known key regulatory genes (Myomaker, MYOD, and MSTN) during pectoral muscle (PM) and thigh muscle (TM) development. The highest expression level of Myomaker occurred from embryonic days E13 to E15 for all breeds, indicating that it was the crucial stage of myoblast fusion. Interestingly, the fast-growing CN showed the highest gene expression level of Myomaker during the crucial stage. The MYOD gene expression at D1 was much higher, implying that MYOD might have an important role after hatching. Histomorphology of PM and TM suggested that the myofibers was largely complete at E17, which was speculated to have occurred because of the expression increase in MSTN and the expression decrease in Myomaker. Our research contributes to lay a foundation for the study of myofiber development during the embryonic period in different chicken breeds.

ABSTRACT: Long noncoding RNAs (lncRNAs) have emerged as important regulators of many biological processes, including embryogenesis and development. To provide a systematic analysis of lncRNAs expressed during chicken embryogenesis, we used Iso-Seq and RNA-Seq to identify potential lncRNAs at embryonic stages from d 1 to d 8 of incubation: sequential stages covering gastrulation, somitogenesis, and organogenesis. The data characterized an expanded landscape of lncRNAs, yielding 45,410 distinct lncRNAs (31,282 genes). Amongst these, a set of 13,141 filtered intergenic lncRNAs (lincRNAs) transcribed from 9803 lincRNA gene loci, of which, 66.5% were novel, were further analyzed. These lincRNAs were found to share many characteristics with mammalian lincRNAs, including relatively short lengths, fewer exons, lower expression levels, and stage-specific expression patterns. Functional studies motivated by “guilt-by-association” associated individual lincRNAs with specific GO functions, providing an important resource for future studies of lincRNA function. Most importantly, a weighted gene co-expression network analysis suggested that genes of the brown module were specifically associated with the day 2 stage. LincRNAs within this module were co-expressed with proteins involved in hematopoiesis and lipid metabolism. This study presents the systematic identification of lincRNAs in developing chicken embryos and will serve as a powerful resource for the study of lincRNA functions.

Abstract Background Marek’s disease virus (MDV) is an oncogenic herpesvirus that can cause T-cell lymphomas in chicken. Long noncoding RNA (lncRNA) is strongly associated with various cancers and many other diseases. In chickens, lncRNAs have not been comprehensively identified. Here, we profiled mRNA and lncRNA repertoires in three groups of spleens from MDV-infected and non-infected chickens, including seven tumorous spleens (TS) from MDV-infected chickens, five spleens from the survivors (SS) without lesions after MDV infection, and five spleens from noninfected chickens (NS), to explore the underlying mechanism of host resistance in Marek’s disease (MD). Results By using a precise lncRNA identification pipeline, we identified 1315 putative lncRNAs and 1166 known lncRNAs in spleen tissue. Genomic features of putative lncRNAs were characterized. Differentially expressed (DE) mRNAs, putative lncRNAs, and known lncRNAs were profiled among three groups. We found that several specific intergroup differentially expressed genes were involved in important biological processes and pathways, including B cell activation and the Wnt signaling pathway; some of these genes were also found to be the hub genes in the co-expression network analyzed by WGCNA. Network analysis depicted both intergenic correlation and correlation between genes and MD traits. Five DE lncRNAs including MSTRG.360.1, MSTRG.6725.1, MSTRG.6754.1, MSTRG.15539.1, and MSTRG.7747.5 strongly correlated with MD-resistant candidate genes, such as IGF-I, CTLA4, HDAC9, SWAP70, CD72, JCHAIN, CXCL12, and CD8B, suggesting that lncRNAs may affect MD resistance and tumorigenesis in chicken spleens through their target genes. Conclusions Our results provide both transcriptomic and epigenetic insights on MD resistance and its pathological mechanism. The comprehensive lncRNA and mRNA transcriptomes in MDV-infected chicken spleens were profiled. Co-expression analysis identified integrated lncRNA-mRNA and gene-gene interaction networks, implying that hub genes or lncRNAs exert critical influence on MD resistance and tumorigenesis.

A rapidly increasing number of reports on dysregulated long intergenic non-coding RNA (lincRNA) expression across numerous types of cancers indicates that aberrant lincRNA expression may be a major contributor to tumorigenesis. Marek’s disease (MD) is a T cell lymphoma of chickens induced by Marek’s disease virus (MDV). Although we have investigated the roles of lincRNAs in bursa tissue of MDV-infected chickens in previous studies, the molecular mechanisms of lincRNA functions in T cells remain poorly understood. In the present study, Linc-GALMD1 was identified from CD4+ T cells and MSB1 cells, and its expression was significantly downregulated in MD-resistant line of birds in response to MDV challenge. Furthermore, loss-of-function experiments indicated that linc-GALMD1 significantly affected the expression of 290 genes in trans. Through integrated analysis of differentially expressed genes (DEGs) induced by MDV and linc-GALMD1, we found that IGLL1 gene expression levels had a positive correlation with the degree of MD infection and could potentially serve as an indicator for clinical diagnosis of MD. Moreover, an interaction between MDV and linc-GALMD1 was also observed. Accordingly, chicken embryonic fibroblast cells were inoculated with MDV with and without the linc-GALMD1 knockdown, and the data showed that linc-GALMD1 could repress MDV gene expression during the course of MDV infection. These findings uncovered a role of linc-GALMD1 as a viral gene regulator and suggested a function of linc-GALMD1 contributing to tumor suppression by coordinating expression of MDV genes and tumor-related genes and regulating immune responses to MDV infection.

Despite the convenience and non-invasiveness of fecal sampling, the fecal microbiota does not fully represent that of the gastrointestinal (GI) tract, and the efficacy of fecal sampling to accurately represent the gut microbiota in birds is poorly understood. In this study, we aim to identify the efficacy of feces as a gut proxy in birds using chickens as a model. We collected 1,026 samples from 206 chickens, including duodenum, jejunum, ileum, cecum, and feces samples, for 16S rRNA amplicon sequencing analyses. In this study, the efficacy of feces as a gut proxy was partitioned to microbial community membership and community structure. Most taxa in the small intestine (84.11–87.28%) and ceca (99.39%) could be identified in feces. Microbial community membership was reflected with a gut anatomic feature, but community structure was not. Excluding shared microbes, the small intestine and ceca contributed 34.12 and 5.83% of the total fecal members, respectively. The composition of Firmicutes members in the small intestine and that of Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria members in the ceca could be well mirrored by the observations in fecal samples (ρ = 0.54–0.71 and 0.71–0.78, respectively, P < 0.001). However, there were few significant correlations for each genus between feces and each of the four gut segments, and these correlations were not high (ρ = −0.2–0.4, P < 0.05) for most genera. Our results suggest that fecal microbial community has a good potential to identify most taxa in the chicken gut and could moderately mirror the microbial structure in the intestine at the microbial population level with phylum specificity. However, it should be interpreted with caution by using feces as a proxy to study associations for microbial structure at individual microorganism level.

Defining the dynamic transcriptome of the early embryo at high resolution would assist greatly in understanding vertebrate development. Here, we describe the dynamic transcription landscape of early chick embryo development using advanced single-molecule long-read isoform sequencing (Iso-Seq) and RNA-Seq technology. Our transcriptomic profiling reflected the time course of chicken embryonic development from day 1 to day 8 of incubation, a period encompassing gastrulation, somitogenesis, and organogenesis. This analysis identified transcriptional isoforms, alternative splicing (AS) events, fusion transcripts, alternative polyadenylation (APA) sites, and novel genes. Our results showed that intron retention (IR) represented the most abundant AS type and displayed distinct features and dynamic modulation during development. Moreover, we constructed a high-resolution expression profile across embryonic development. Our combined expression dataset correlates distinct gene clusters with specific morphological changes, and provides the first framework for the molecular basis of early chicken embryogenesis. Analysis of gene expression in the developing chicken embryo highlighted the dynamic nature and complexity of the chicken transcriptome and demonstrated that dramatically increased IR events are associated with distinct gene sets.

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.

Abstract: Damaged eggshells result in losses of eggs. The genetic mechanism of variable eggshell strength is still unclear. The current study was conducted to verify whether the eggshell calcification related genes, CALB1, SPP1, DMP4, BMP2 and SLIT2, were associated with eggshell mechanical property. For this purpose quantitative PCR (q-PCR) analysis was performed to detect gene expression between two groups of hens laying strong and weak eggs. The hens were selected from 360 White Leghorn layers at 60 wk to ensure that the strong and weak eggs differed significantly in breaking strength but not in eggshell thickness and weight. Using this special strong/weak eggshell model, we found that the expression of CALB1 in the uterus of strong shell group was about 3-fold higher (P

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.

Cytochrome P450 (CYP) superfamily enzymes are broadly involved in a variety of physiological and toxicological processes. However, genome-wide analysis of this superfamily has never been investigated in the chicken genome. In this study, genome-wide analyses identified 45 chicken CYPs (cCYPs) from the chicken genome, and their classification and evolutionary relationships were investigated by phylogenetic, conserved protein motif, and gene structure analyses. The comprehensive evolutionary data revealed several remarkable characteristics of cCYPs, including the highly divergent and rapid evolution of the cCYPs, and the loss of cCYP2AF in the chicken genome. Furthermore, the cCYP expression profile was investigated by RNA-sequencing. The differential expression of cCYPs in developing embryos revealed the involvement of cCYPs in embryonic development. The significantly regulated cCYPs suggested its potential role in hepatic metabolism. Additionally, 11 cCYPs, including cCYP2AC1, cCYP2C23a, and cCYP2C23b, were identified as estrogen-responsive genes, which indicates that these cCYPs are involved in the estrogen-signaling pathway. Meanwhile, an expression profile analysis highlights the divergent role of different cCYPs. These data expand our view of the phylogeny and evolution of cCYPs, provide evolutionary insight, and can help elucidate the roles of cCYPs in physiological and toxicological processes in chicken.

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.

Fadrozole, an aromatase inhibitor, can masculinize genetic female chickens and high-dose decreases the hatchability. Therefore, it is important to study the growth and development of sex-reversed females after hatch. Chick embryos from a population of CAU3 egg-type were treated with different concentrations of Fadrozole prior to the sexual differentiation at E3.0 (st18). At hatch, the phenotypic sex and genetic sex were identified by vent sexing and genetic diagnosis with CHD1, respectively. Body weight and shank length of sex reversal were tested at 8 and 20 wk, respectively. Testicular development, oviduct and ovarian degeneration were observed and serum concentration of estradiol and testosterone were tested with radioimmunoassay (RIA) at 30 wk. The results showed that body weight and shank length of sex-reversed females were not significantly different between low-dose groups (0.1, 0.3, and 0.5 mg for F1, F2, and F3, respectively) and high-dose groups (1.0 and 1.3 mg for F4 and F5, respectively) (P>0.05). Left and right testes or ovotestes in F2, F3, F4, and F5 groups were heavier than that of in F1 group (P

The environmental stimuli experienced by a female can influence phenotypes and gene expression in the subsequent generations. We used a specifically designed domesticated-bird model to examine the transgenerational transmission of maternal stimulus exposure, a phenomenon that has been observed but has not been understood in noninbred animals. We subjected parental generation [filial (F)0] hens to viral- or bacterial-like stimulation after artificial insemination. Subsequent filial generations F1 and F2 transmitted growth or fertility variations without further stimulation in contrast to the controls. The whole-genome bisulfite sequence and next-generation mRNA sequencing of peripheral blood lymphocytes (PBLs) from the F1 generation revealed DNA methylome and transcriptome differences in the F1 polyriboinosinic:polyribocytidylic [poly(I:C)] acid or LPS offspring, compared with the F1 controls. In the F1 offspring, DNA methylation changes induced by maternal immune stimulation may have contributed to transcriptional variation. Pathways analysis indicated that the metabolic processes of xenobiotics and drug metabolism pathways, as well as reproduction-related pathways, were involved in the transgenerational transmission of maternal stimulatory experience. Furthermore, LPS-induced transcriptional transmission may have contributed to subfertility, as indicated by the results of comparative analysis between the transcriptomes of spleen tissues across the F0 and F1 generations, as well as the correlative analysis between the transcriptome and reproductive phenotypes. Our findings provide a framework for determining the mechanisms by which maternal stimulatory factors can be inherited transgenerationally with respect to growth, fertility, DNA methylation, and transcriptional levels in outbred animals. [ABSTRACT FROM AUTHOR]

Background: Copy number variation (CNV) is a major source of genome polymorphism that directly contributes to phenotypic variation such as resistance to infectious diseases. Lines 63 and 72 are two highly inbred experimental chicken lines that differ greatly in susceptibility to Marek's disease (MD), and have been used extensively in efforts to identify the genetic and molecular basis for genetic resistance to MD. Using next generation sequencing, we present a genome-wide assessment of CNVs that are potentially associated with genetic resistance to MD. Methods: Three chickens randomly selected from each line were sequenced to an average depth of 20×. Two popular software, CNVnator and Pindel, were used to call genomic CNVs separately. The results were combined to obtain a union set of genomic CNVs in the two chicken lines. Results: A total of 5,680 CNV regions (CNVRs) were identified after merging the two datasets, of which 1,546 and 1,866 were specific to the MD resistant or susceptible line, respectively. Over half of the line-specific CNVRs were shared by 2 or more chickens, reflecting the reduced diversity in both inbred lines. The CNVRs fixed in the susceptible lines were significantly enriched in genes involved in MAPK signaling pathway. We also found 67 CNVRs overlapping with 62 genes previously shown to be strong candidates of the underlying genes responsible for the susceptibility to MD. Conclusions: Our findings provide new insights into the genetic architecture of the two chicken lines and additional evidence that MAPK signaling pathway may play an important role in host response to MD virus infection. The rich source of line-specific CNVs is valuable for future disease-related association studies in the two chicken lines. [ABSTRACT FROM AUTHOR]

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