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Abstract Egg-laying performance is of great economic importance in poultry, but the underlying genetic mechanisms are still elusive. In this work, we conduct a multi-omics and multi-tissue integrative study in hens with distinct egg production, to detect the hub candidate genes and construct hub molecular networks contributing to egg-laying phenotypic differences. We identifiy three hub candidate genes as egg-laying facilitators: TFPI2, which promotes the GnRH secretion in hypothalamic neuron cells; CAMK2D, which promotes the FSHβ and LHβ secretion in pituitary cells; and OSTN, which promotes granulosa cell proliferation and the synthesis of sex steroid hormones. We reveal key endocrine factors involving egg production by inter-tissue crosstalk analysis, and demonstrate that both a hepatokine, APOA4, and an adipokine, ANGPTL2, could increase egg production by inter-tissue communication with hypothalamic-pituitary-ovarian axis. Together, These results reveal the molecular mechanisms of multi-tissue coordinative regulation of chicken egg-laying performance and provide key insights to avian reproductive regulation.

Abstract Breast muscle growth rate and intramuscular fat (IMF) content show apparent differences between fast-growing broilers and slow-growing indigenous chickens. However, the underlying genetic basis of these phenotypic characteristics remains elusive. In this study, we investigate the dynamic alterations of three-dimensional genome architecture and chromatin accessibility in breast muscle across four key developmental stages from embryo to starter chick in Arbor Acres (AA) broilers and Yufen (YF) indigenous chickens. The limited breed-specifically up-regulated genes (Bup-DEGs) are embedded in breed-specific A compartment, while a majority of the Bup-DEGs involving myogenesis and adipogenesis are regulated by the breed-specific TAD reprogramming. Chromatin loops allow distal accessible regions to interact with myogenic genes, and those loops share an extremely low similarity between chicken with different growth rate. Moreover, AA-specific loop interactions promote the expression of 40 Bup-DEGs, such as IGF1, which contributes to myofiber hypertrophy. YF-specific loop interactions or distal accessible regions lead to increased expression of 5 Bup-DEGs, including PIGO, PEMT, DHCR7, TMEM38B, and DHDH, which contribute to IMF deposition. These results help elucidate the regulation of breast muscle growth and IMF deposition in chickens.

Abstract Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.

C1q/tumor necrosis factor-related protein 6 (C1QTNF6) is a newly discovered adiponectin analog that plays a significant role in female reproduction. However, its expression and function in chickens remain unclear. In this study, the full-length coding sequence (CDS) of chicken C1QTNF6 was cloned from adult chicken ovary. We demonstrated that the putative C1QTNF6 protein shares a highly conserved amino acid sequence with known bird homologs. Using quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH), we observed that C1QTNF6 is widely expressed in adult chicken follicle tissues, predominantly in granulosa cells (GCs). In vitro, stimulation with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) significantly increased the expression of C1QTNF6 in chicken preovulatory granulosa cells (PoGCs) and prehierarchical granulosa cells (PhGCs), respectively. Overexpression and knockdown experiments in granulosa cells revealed that C1QTNF6 affects the proliferation and apoptosis of granulosa cells, reducing the expression of steroid hormone synthesis-related genes (STAR, Cyp11a1, Cyp19a1, and 3B-HSD) and hormones (progesterone and estrogen). In granulosa cells (GCs), co-treatment with C1QTNF6 and AdipoRon (an activator of adiponectin receptors) showed that AdipoRon promotes progesterone secretion, while C1QTNF6 inhibits AdipoR1 (adiponectin receptor 1) expression. There was no synergistic effect between C1QTNF6 and AdipoRon in steroid hormone production. Collectively, these findings suggest that C1QTNF6 may be a candidate gene for regulating follicular development and reproductive performance in chickens.

ABSTRACT: Though it is well known that insulin-like growth factor (IGF) binding protein 7 (IGFBP7) plays an important role in myogenesis and adipogenesis in mammals, its impact on the proliferation, differentiation, and lipid deposition in chicken primary myoblasts (CPM) and intramuscular preadipocytes remains unexplored. In the present study, we firstly examined the correlation between SNPs within the genomic sequence of the IGFBP7 gene and carcass and blood chemical traits in a F2 resource population by genetic association analysis, and found that a significant correlation between the SNP (4_49499525) located in the intron region of IGFBP7 and serum high-density lipoproteins (HDL). We then examined the expression patterns of IGFBP7 across different stages of proliferation and differentiation in CPMs and intramuscular preadipocytes via qPCR, and explored the biological functions of IGFBP7 through gain- and loss-of-function experiments and a range of techniques including qPCR, CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and Oil Red O staining to detect the proliferation, differentiation, and lipid deposition in CPMs and intramuscular preadipocytes. We ascertained that the expression levels of the IGFBP7 gene increased as cell differentiation progresses in CPMs and intramuscular preadipocytes, and that IGFBP7 promotes the proliferation and differentiation of these cells, as well as facilitates intracellular lipid deposition. Furthermore, we investigated the regulatory mechanism of IGFBP7 expression by using co-transfection strategy and dual-luciferase reporter assay, and discovered that the myogenic transcription factors (MRF), myoblast determination factor (MyoD) and myogenin (MyoG), along with the adipocyte-specific transcription factor (TF) CCAAT/enhancer-binding protein α (C/EBPα), can bind to the core transcription activation region of the IGFBP7 promoter located 500 bp upstream from the transcription start site, thereby promoting IGFBP7 transcription and expression. Taken together, our study underscores the role of IGFBP7 as a positive regulator for myogenesis and adipogenesis, while also elucidating the functional and transcriptional regulatory mechanisms of IGFBP7 in chicken skeletal muscle development and intramuscular adipogenesis.

ABSTRACT: The economic losses incurred due to reduced muscle pigmentation highlight the crucial role of melanin-based coloration in the meat of black-bone chickens. Melanogenesis in the breast muscle of black-bone chickens is currently poorly understood in terms of molecular mechanisms. This study employed whole-transcriptome sequencing to analyze black and white breast muscle samples from black-bone chickens, leading to the identification of 367 differentially expressed (DE) mRNAs, 48 DElncRNAs, 104 DEcircRNAs, and 112 DEmiRNAs involved in melanin deposition. Based on these findings, a competitive endogenous RNA (ceRNA) network was developed to better understand the complex mechanisms of melanin deposition. Furthermore, our analysis revealed key DEmRNAs (TYR, DCT, EDNRB, MLPH and OCA2) regulated by DEmiRNAs (gga-miR-140-5p, gga-miR-1682, gga-miR-3529, gga-miR-499-3p, novel-m0012-3p, gga-miR-200b-5p, gga-miR-203a, gga-miR-6651-5p, gga-miR-7455-3p, gga-miR-31-5p, miR-140-x, miR-455-x, novel-m0065-3p, gga-miR-29b-1-5p, miR-455-y, novel-m0085-3p, and gga-miR-196-1-3p). These DEmiRNAs competitively interacted with DElncRNAs including MSTRG.2609.2, MSTRG.4185.1, LOC112530666, LOC112533366, LOC771030, LOC107054724, LOC121107411, LOC100859072, LOC101750037, LOC121108550, LOC121109224, LOC121110876, and LOC101749016, as well as DEcircRNAs, such as novel_circ_000158, novel_circ_000623, novel_001518, and novel_circ_003596. The findings from this study provide insight into the mechanisms that regulate lncRNA, circRNA, miRNA, and mRNA expression in chicken melanin deposition.

ABSTRACT: During myogenesis and regeneration, the proliferation and differentiation of myoblasts play key regulatory roles and may be regulated by many genes. In this study, we analyzed the transcriptomic data of chicken primary myoblasts at different periods of proliferation and differentiation with protein‒protein interaction network, and the results indicated that there was an interaction between cyclin-dependent kinase 1 (CDK1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Previous studies in mammals have a role for RRM2 in skeletal muscle development as well as cell growth, but the role of RRM2 in chicken is unclear. In this study, we investigated the effects of RRM2 on skeletal muscle development and regeneration in chickens in vitro and in vivo. The interaction between RRM2 and CDK1 was initially identified by co-immunoprecipitation and mass spectrometry. Through a dual luciferase reporter assay and quantitative real-time PCR, we identified the core promoter region of RRM2, which is regulated by the SP1 transcription factor. In this study, through cell counting kit-8 assays, 5-ethynyl-2′-deoxyuridine incorporation assays, flow cytometry, immunofluorescence staining, and Western blot analysis, we demonstrated that RRM2 promoted the proliferation and inhibited the differentiation of myoblasts. In vivo studies showed that RRM2 reduced the diameter of muscle fibers and slowed skeletal muscle regeneration. In conclusion, these data provide preliminary insights into the biological functions of RRM2 in chicken muscle development and skeletal muscle regeneration.

Abstract Background Modern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler. Results Two time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2. TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts. Conclusions For the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

Abstract Background Intramuscular fat (IMF) content is the major indicator for evaluating chicken meat quality due to its positive correlation with tenderness, juiciness, and flavor. An increasing number of studies are focusing on the functions of microRNAs (miRNAs) in intramuscular adipocyte differentiation. However, little is known about the association of miR-128-3p with intramuscular adipocyte differentiation. Our previous RNA-seq results indicated that miR-128-3p was differentially expressed at different periods in chicken intramuscular adipocytes, revealing a possible association with intramuscular adipogenesis. The purpose of this research was to investigate the biological functions and regulatory mechanism of miR-128-3p in chicken intramuscular adipogenesis. Results The results of a series of assays confirmed that miR-128-3p could promote the proliferation and inhibit the differentiation of intramuscular adipocytes. A total of 223 and 1,050 differentially expressed genes (DEGs) were identified in the mimic treatment group and inhibitor treatment group, respectively, compared with the control group. Functional enrichment analysis revealed that the DEGs were involved in lipid metabolism-related pathways, such as the MAPK and TGF-β signaling pathways. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DEGs, such as FDPS, GGT5, TMEM37, and ASL2. The luciferase assay results showed that miR-128-3p targeted the 3’ UTR of FDPS. The results of subsequent functional assays demonstrated that miR-128-3p acted as an inhibitor of intramuscular adipocyte differentiation by targeting FDPS. Conclusion miR-128-3p inhibits chicken intramuscular adipocyte differentiation by downregulating FDPS. Our findings provide a theoretical basis for the study of lipid metabolism and reveal a potential target for molecular breeding to improve meat quality.

ABSTRACT: miR-19b-3p is reported to undertake various biological role, while its function and action mechanism in chicken hepatic lipid metabolism is unclear. Conservation analysis and tissue expression pattern of miR-19b-3p and its target gene were evaluated, respectively. Dual luciferase reporter system and Western blot technologies were adopted to validate miR-19b-3p target gene. Overexpression and knockdown assays were done to explore the biological functions of miR-19b-3p and target gene in Leghorn Male Hepatoma cell line (LMH). Regulatory approaches of estrogen on miR-19b-3p and target gene expressions are analyzed through site-directed mutation combined with estrogen receptors antagonist treatment assays. The results showed that chicken miR-19b-3p mature sequences are highly conserved among Capra hircus, Columba livia, Rattus norvegicus, Mus musculus, Cricetulus griseus, Danio rerio, Danio novaehollandiae, Orycodylus porosus, Crocodylus porosus, Gadus morhua, and widely expressed in lung, ovary, spleen, duodenum, kidney, heart, liver, leg muscle, and pectoral muscle tissues. miR-19b-3p could significantly increase intracellular triglyceride (TG) content and decrease intracellular cholesterol (TC) content via targeting methylsterol monooxygenase 1 (MSMO1) and elongase of very long chain fatty acids 5 (ELOVL5), which are highly conserved among species, in both mRNA and protein levels. Estrogen could inhibit miR-19b-3p expression, but directly promoted MSMO1 transcription via estrogen receptor α (ERα) and indirectly regulated ELOVL5 expression at the transcription level. Meanwhile, estrogen could also upregulate MSMO1 and ELOVL5 expression through inhibiting miR-19b-3p expression at the post-transcription level. Taken together, these results highlight the role and regulatory mechanism of miR-19b-3p in hepatic lipid metabolism in chicken, and might produce useful comparative information for human obesity studies and biomedical research.

Abstract Background Serum biochemical indicators are often regarded as direct reflections of animal metabolism and health. The molecular mechanisms underlying serum biochemical indicators metabolism of chicken (Gallus Gallus) have not been elucidated. Herein, we performed a genome-wide association study (GWAS) to identify the variation associated with serum biochemical indicators. The aim of this research was to broaden the understanding of the serum biochemical indicators in chickens. Results A GWAS of serum biochemical indicators was carried out on 734 samples from an F2 Gushi× Anka chicken population. All chickens were genotyped by sequencing, 734 chickens and 321,314 variants were obtained after quality control. Based on these variants, a total of 236 single-nucleotide polymorphisms (SNPs) on 9 chicken chromosomes (GGAs) were identified to be significantly (-log10(P) > 5.72) associated with eight of seventeen serum biochemical indicators. Ten novel quantitative trait locis (QTLs) were identified for the 8 serum biochemical indicator traits of the F2 population. Literature mining revealed that the ALPL, BCHE, GGT2/GGT5 genes at loci GGA24, GGA9 and GGA15 might affect the alkaline phosphatase (AKP), cholinesterase (CHE) and γ-glutamyl transpeptidase (GGT) traits, respectively. Conclusion The findings of the present study may contribute to a better understanding of the molecular mechanisms of chicken serum biochemical indicator regulation and provide a theoretical basis for chicken breeding programs.

ABSTRACT: The reproductive performance of breeder roosters has significant economic importance in the poultry industry. Breeder roosters have severely reduced semen quality with age and will be at risk of culling in the following years. In order to extend the use of breeder roosters, we drew on the induced molting model of hens and selected 35 Houdan roosters aged 50 wk for induced molting. By comparing the body weight, testicular weight, semen quality, and reproductive performance before and after induced molting, we found that induced molting could restore the body weight and testicular weight to the levels before molting (P > 0.05). At the same time, it significantly improved sperm motility (P < 0.05) and also improved reproductive performance such as fertilization rate and hatching rate. To further reveal the mechanism underlying the effects of induced molting on semen quality and reproductive performance in aged Houdan roosters, we collected testes from 3 periods: 1 d before fasting (F0), 15 d after fasting (F15), and 32 d after recovery feeding (R32) for transcriptome sequencing analysis. A total of 5,671 genes were detected in F0, F15, and R32, and trend analysis of the 5,671 differential genes showed 2 significant trends (profile 5 and profile 2). KEGG enrichment analysis of the genes in the 2 profiles, revealed significantly enriched pathway regulation of actin cytoskeleton. In the regulation of actin cytoskeleton pathway, we found a protein kinase gene (SRC) and a senescence gene (ROCK2). SRC was highly expressed at F15, leading to the phosphorylation of key substrates, which in turn disrupted the Sertoli cell spermatid connection and the spermiogenesis process, resulting in no mature spermatozoa produced from F15, SRC expression was inhibited at R32, the expression level was reduced, and mature spermatozoa reappeared. The senescence gene ROCK2 was highly expressed at F15 compared to F0 and R32, which may have been responsible for inducing senescence atrophy in the testes.

Abstract Background In molecular biology studies, the selection of optimal reference genes is of vital importance for accurately quantifying gene expression. The purpose of the present study was to screen the most stable reference genes in different muscle tissues of New Zealand white rabbits and Yufeng yellow rabbits. Methods and results Results indicated that the most stable reference genes in the muscle tissues of New Zealand white rabbits were HPRT1, ACTB and PPIC, while HPRT1, PPIC, and RPL13A were the most stable reference genes in muscle tissues of Yufeng yellow rabbits. However, in the longissimus dorsi muscle and the abdominal wall muscle of both varieties, the most stable reference genes were HPRT1, RPL13A, and SDHA. In the quadriceps femoris muscle, the most stable reference genes were ACTB, HPRT1, and SDHA. Furthermore, the relative abundance of MYOG, MYH3 and MSTN was used to confirm the suitability and reliability of the selected most stable reference genes and the most unstable reference gene. Results revealed the same expression patterns of these myogenic genes when normalized according to the most stable genes, while normalization against the unstable reference gene altered the observed expression patterns. Conclusions Taken together, our results demonstrated that the most stable reference genes varied among different muscle tissues and different breeds of rabbits. However, HPRT1, PPIC and SDHA presented high stability among all examined reference genes; thus, the combined analysis of HPRT1/ PPIC/ SDHA gene provides the best reference for RT-qPCR in muscle tissues of New Zealand white rabbits and Yufeng yellow rabbits, while HPRT1 is a better choice than other reference genes when using a single reference gene to assess target gene expression. Our results provide basic data for better measuring target gene expression profiles in muscle tissues of rabbits.

Abstract Background Elongases of very long chain fatty acids (ELOVLs), a family of first rate-limiting enzymes in the synthesis of long-chain fatty acids, play an essential role in the biosynthesis of complex lipids. Disrupting any of ELOVLs affects normal growth and development in mammals. Genetic variations in ELOVLs are associated with backfat or intramuscular fatty acid composition in livestock. However, the effects of ELOVL gene family on breeding selection and lipid deposition in different tissues are still unknown in chickens. Results Genetic variation patterns and genetic associations analysis showed that the genetic variations of ELOVL genes were contributed to breeding selection of commercial varieties in chicken, and 14 SNPs in ELOVL2-6 were associated with body weight, carcass or fat deposition traits. Especially, one SNP rs17631638T > C in the promoter of ELOVL3 was associated with intramuscular fat content (IMF), and its allele frequency was significantly higher in native and layer breeds compared to that in commercial broiler breeds. Quantitative real-time PCR (qRT-PCR) determined that the ELOVL3 expressions in pectoralis were affected by the genotypes of rs17631638T > C. In addition, the transcription levels of ELOVL genes except ELOVL5 were regulated by estrogen in chicken liver and hypothalamus with different regulatory pathways. The expression levels of ELOVL1-6 in hypothalamus, liver, abdominal fat and pectoralis were correlated with abdominal fat weight, abdominal fat percentage, liver lipid content and IMF. Noteworthily, expression of ELOVL3 in pectoralis was highly positively correlated with IMF and glycerophospholipid molecules, including phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol and phospholipids inositol, rich in ω-3 and ω-6 long-chain unsaturated fatty acids, suggesting ELOVL3 could contribute to intramuscular fat deposition by increasing the proportion of long-chain unsaturated glycerophospholipid molecules in pectoralis. Conclusions In summary, we demonstrated the genetic contribution of ELOVL gene family to breeding selection for specialized varieties, and revealed the expression regulation of ELOVL genes and their potential roles in regulating lipid deposition in different tissues. This study provides new insights into understanding the functions of ELOVL family on avian growth and lipid deposition in different tissues and the genetic variation in ELOVL3 may aid the marker-assisted selection of meat quality in chicken.

ABSTRACT: Induced molting (IM) can restore the laying rate of aged laying hens to the peak level of laying and rejuvenate ovarian function for the second laying cycle. To explore the mechanism of ovarian function remodeling during IM in laying hens, in this study, ninety 71-wk-old laying lady hens with 60% laying rate and uniform weight were selected for molting induction by fasting. Samples (serum and fresh ovarian tissue) were collected on the day before fasting (F0), the 3rd and 16th days of fasting (F3, F16), and the 6th, 15th, 32nd days of refeeding (R6, R15, and R32), and the number of follicles in each period was counted. Then, the reproductive hormone levels in serum and antioxidant levels in ovarian tissues were detected at different stages, and the gene expression of the KIT-PI3K-PTEN-AKT pathway and GDF-9 in ovaries was measured by qRT-PCR. The results showed that the laying rate increased rapidly after refeeding and returned to the prefasting level by R32. At F16 and R6, the number of mature follicles significantly decreased; the number of primary and secondary follicles significantly increased; the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P4) in serum decreased; the relative expression of KIT, PI3K, AKT, and GDF-9 significantly increased; and that of PTEN significantly decreased. At R15 and R32, except for GDF-9, which maintained a high expression state, other indicators showed opposing trends to those observed at F16 and R6. In conclusion, IM activated the KIT-PI3K-PTEN-AKT signaling pathway and promoted the activation of primordial follicles during the fasting period and early resumption of feeding; gonadotropin secretion increased gradually, which promoted the rapid development of primary and secondary follicles to mature follicles and ovulation. This study explained the mechanism of ovarian function remodeling in the process of IM and provided a theoretical basis for improving the ovarian function of laying hens and optimizing the IM program.

ABSTRACT: Valgus-varus deformity (VVD) is a common leg disease in commercial broilers, which seriously affects animal welfare and causes economic losses. Up to now, most of the studies on VVD have been on skeleton, whereas there are fewer studies on VVD muscle. In this study, carcass composition and meat quality of 35-day-old normal and VVD Cobb broilers assess the effect of VVD on broiler growth. Molecular biology, morphology, and RNA sequencing (RNA-seq) were used to study the difference between normal and VVD gastrocnemius muscle. In comparison with the normal broilers, the breast muscle and leg muscle of the VVD broilers had lower shear force, notably lower crude protein, lower water content, cooking loss, and deeper meat color (P < 0.05). The morphological results showed that the weight of skeletal muscle was significantly higher in the normal broilers than that in the VVD broilers (P < 0.01), the diameter and area of myofibrils in the affected VVD were smaller than in the normal broilers (P < 0.01). Quantitative real-time PCR (qPCR) of gastrocnemius muscle revealed that the expression of myasthenic marker genes, fast myofiber marker genes, and apoptosis-related factors were significantly higher in the VVD broilers than in the normal broilers (P < 0.01). In total, 736 differentially expressed genes (DEGs) were identified firstly in the normal and VVD leg muscle by RNA-seq. Gene ontology (GO) enrichment indicated that these DEGs were mainly involved in the multicellular organismal process and anatomical structure development. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs are significantly enriched in proteasome. Protein interaction analysis obtained that DEGs with high interaction were proteasome-related coding genes and ubiquitin-related genes, these DEGs were closely associated with muscle atrophy. These show that VVD has an adverse effect on growth characteristics, slaughter characteristics, and meat quality in broilers, which may cause leg muscle atrophy. This study provides some reference values and basis for studying the pathogenesis of VVD in broilers.

ABSTRACT: Columbian coloration patterns in plumage are widespread phenomena in several standard breeds of poultry, such as the Columbian Plymouth Rock chicken. H line chicken plumage is generally a pure white except in the hackle, wing, and tail plumage, which coloration is very similar to the Columbian plumage pattern, but with the barring substituting for the black vertical striping. Thus, we refer to this plumage coloration as “sub-Columbian” pattern. However, the genetic basis of this phenotype remains unknown. Here, a F3 cross population between yellow plumage roosters and sub-Columbian plumage hens was constructed, for verifying sub-Columbian plumage was sex-linked dominant inheritance. To identify the candidate regions, F2 generation sub-Columbian plumage hens and yellow plumage hens with their parental lines were used for BSA-seq, and sub-Columbian plumage genes were mapped to a 10.46 Mb interval on chromosome Z. Remarkably, by transcriptome analysis of the neck and wing tip follicle tissues of the 2 plumage colors, we demonstrated that within the interval, only 1 gene, SLC45A2 expressed significant differently (P < 0.05). Through KASP, we identified L347M and A10331272T in solute carrier family 45 member 2 (SLC45A2), and B2 haplotype of cyclin-dependent kinase inhibitor 2A (CDKN2A), showed near complete association with the phenotype. Eventually, we designed a hybridization experiment for verifying the locus of sub-Columbian plumage, which is inherited through Z-linked dominant inheritance and is controlled by SLC45A2 and CDKN2A.

ABSTRACT: The insulin-like growth factor (IGF) system plays an indispensable role in embryonic and postnatal development in mammals. However, the effects of the system on growth, carcass, and egg-laying traits, and diversified selection have not been systematically studied in chickens. In the present study, firstly the composition and gene structures of the chicken IGF system were investigated using phylogenetic tree and conserved synteny analysis. Then the effects of the genetic variations in the IGF system genes on breeding of specialized varieties were explored by principal component analysis. In addition, the spatiotemporal expression properties of the genes in this system were analyzed by RT-qPCR and the functions of the genes in egg production performance and growth were explored by association study. Moreover, the effects of IGF-binding proteins 3 (IGFBP3) on skeletal muscle development in chicken were investigated by cell cycle analysis, 5-ethynyl-2′-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays. The results showed that the chicken IGF system included 13 members which could be classified into 3 groups based on their amino acid sequences: IGF binding proteins 1 to 5 and 7 (IGFBP1–5 and 7) belonged to the first group; IGF 1 and 2 (IGF1 and IGF2), and IGF 1 and 2 receptor (IGF1R and IGF2R) belonged to the second group; and IGF2 binding proteins 1–3 (IGF2BP1–3) belonged to the third group. The IGF2BP1 and 3, and IGFBP2, 3, and 7 genes likely contributed more to the formation of both the specialized meat-type and egg-type lines, whereas IGFBP1 and 5 likely contributed more to the formation of the egg-type lines. The SNPs in the IGF2BP3 and IGFBP2 and 5 genes were significantly associated with egg number, and SNPs in the IGFBP3 promoter region were significantly associated with body weight, breast muscle weight and leg muscle weight. The IGFBP3 inhibited proliferation but promoted differentiation of chicken primary myoblasts (CPMs). These results provide insights into the roles of the IGF system in the diversified selection of chickens. The SNPs associated with egg-laying performance, growth, and carcass traits could be used as genetic markers for breeding selection of chickens in the future.

Induced molting is a common method to obtain a new life in laying hens, in which periodic changes in feathers are the prominent feature. Nevertheless, its precise molecular mechanism remains unclear. In this study, feather and hair follicle samples were collected during fasting-induced physiological remodeling for hematoxylin–eosin staining, hormone changes and follicle traits, and transcriptome sequencing. Feather shedding was observed in F13 to R25, while newborns were observed in R3 to R32. Triiodothyronine and tetraiodothyronine were significantly elevated during feather shedding. The calcium content was significantly higher, and the ash content was significantly lower after the changeover. The determination of hair follicle traits revealed an increasing trend in pore density and a decrease in pore diameter after the resumption of feeding. According to RNA-seq results, several core genes were identified, including DSP, CDH1, PKP1, and PPCKB, which may have an impact on hair follicle growth. The focus was to discover that starvation may trigger changes in thyroid hormones, which in turn regulate feather molting through thyroid hormone synthesis, calcium signaling, and thyroid hormone signaling pathways. These data provide a valuable resource for the analysis of the molecular mechanisms underlying the cyclical growth of hair follicles in the skin during induced molting.

Abstract Background Molecular breeding accelerates the speed of animal breeding. Screening molecular markers that can affect economic traits through genome-wide association studies (GWAS) can provide a theoretical basis for molecular breeding. At present, a large number of molecular markers have been screened in poultry research, but few reports on how molecular markers affect economic traits exist. It is particularly important to reveal the action mechanisms of molecular markers, which can provide more accurate information for molecular breeding. Results The aim of this study was to investigate the relationships between two indels (NUDT15-indel-2777 and NUDT15-indel-1673) in the promoter region of NUDT15 and growth and carcass traits in chickens and to explore the regulatory mechanism of NUDT15. Significant differences were found in genotype and allele frequencies among commercial broilers, commercial laying hens and dual-purpose chickens. The results of association analyses showed that these two indel loci could significantly affect growth traits, such as body weight, and carcass traits. Tissue expression profiling at E12 showed that the expression of NUDT15 was significantly higher in skeletal muscle, and time-expression profiling of leg muscle showed that the expression of NUDT15 in myoblasts was significantly higher in the E10 and E12 proliferation stages than in other stages. Promoter activity analysis showed that pro-1673-I and pro-1673-D significantly inhibited promoter activity, and the promoter activity of pro-1673-D was significantly lower than that of pro-1673-I. In addition, when NUDT15 was overexpressed or underwent interference in chicken primary myoblasts (CPMs), NUDT15 could inhibit the proliferation of CPMs. Conclusion The results suggest that the studied indels in the promoter region of NUDT15 may regulate the proliferation of CPMs by affecting NUDT15 expression, ultimately affecting the growth and carcass traits of chickens. These indel polymorphisms may be used together as molecular markers for improving economic traits in chickens.

Abstract Background Valgus-varus deformity (VVD) is a lateral or middle deviation of the tibiotarsus or tarsometatarsus, which is associated with compromised growth, worse bone quality and abnormal changes in serum indicators in broilers. To investigate the genetic basis of VVD, a genome wide association study (GWAS) was performed to identify candidate genes and pathways that are responsible for VVD leg disease, serum indicators and growth performance in broilers. Results In total, VVD phenotype, seven serum indicators and three growth traits were measured for 126 VVD broilers (case group) and 122 sound broilers (control group) based on a high throughput genome wide genotyping-by-sequencing (GBS) method. After quality control 233 samples (113 sound broilers and 120 VVD birds) and 256,599 single nucleotide polymorphisms (SNPs) markers were used for further analysis. As a result, a total of 5 SNPs were detected suggestively significantly associated with VVD and 70 candidate genes were identified that included or adjacent to these significant SNPs. In addition, 43 SNPs located on Chr24 (0.22 Mb - 1.79 Mb) were genome-wide significantly associated with serum alkaline phosphatase (ALP) and 38 candidate genes were identified. Functional enrichment analysis showed that these genes are involved in two Gene Ontology (GO) terms related to bone development (cartilage development and cartilage condensation) and two pathways related to skeletal development (Toll−like receptor signaling pathway and p53 signaling pathway). BARX2 (BARX homeobox 2) and Panx3 (Pannexin 3) related to skeleton diseases and bone quality were obtained according to functional analysis. According to the integration of GWAS with transcriptome analysis, HYLS1 (HYLS1 centriolar and ciliogenesis associated) was an important susceptibility gene. Conclusions The results provide some reference for understanding the relationship between metabolic mechanism of ALP and pathogenesis of VVD, which will provide a theoretical basis for disease-resistant breeding of chicken leg soundness.

ABSTRACT: The insulin-induced genes (INSIG1 and INSIG2) have been demonstrated to play a vital role in regulating lipid metabolism in mammals, however the function and regulation mechanism of them remains unknown in poultry. In this study, firstly the phylogenetic trees of INSIGs among various species were constructed and their subcellular locations were mapped in chicken LMH. Then the spatiotemporal expression profiles, over-expression and knockdown assays of chicken INSIGs were conducted. Furthermore, conservation of potential miRNA binding sites in INSIGs among species were analyzed, and the miRNA biological function and regulatory role were verified. The results showed that chicken INSIGs located in cellular endoplasmic reticulum, and were originated from the common ancestors of their mammalian counterparts. The INSIGs were widely expressed in all detected tissues, and their expression levels in the liver of chicken at 30 wk were significantly higher than that at 20 wk (P < 0.01). Over-expression of INSIGs led no significant increase in mRNA abundance of lipid metabolism-related genes and the contents of triacylglycerol (TG) and cholesterol (TC) in LMH cells. Knockdown of INSIG1 led to the decreased expressions of ACSL1, MTTP-L, ApoB, ApoVLDLII genes and TG, TC contents (P < 0.05). Knockdown of INSIG2 could significantly decrease the contents of TG and TC, and expressions of key genes related to the lipid metabolism (P < 0.05). Moreover, INSIG1 was directly targeted by both miR-130b-3p and miR-218-5p, and INSIG2 was directly targeted by miR-130b-3p. MiR-130b-3p mimic and miR-218-5p mimic treatment could significant decrease the mRNA and protein levels of INSIGs, mRNA levels of genes related to lipid metabolism, and the contents of TG and TC in LMH cells. The inhibition of miR-130b-3p and miR-218-5p on TG and TC contents could be restored by the overexpression of INSIGs, respectively. No significant alteration in expressions of sterol regulatory element binding protein (SREBPs) and SREBP cleavage-activating protein (SCAP) were observed when INSIGs were over-expressed. SCAP was down-regulated when INSIG1 was knocked down, while SREBP1 was down-regulated when INSIG2 was knocked down. Taken together, these results highlight the role of INSIG1 and INSIG2 in lipid metabolism and their regulatory mechanism in chicken.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a vital secreted factor that promotes the occurrence of obesity in mammals. However, the effects of GPNMB on abdominal fat deposition is still unknown in chickens. In this study, we looked into the genetic and expression association of GPNMB gene with abdominal fat traits in chicken, and found that a genetic variation rs31126482 in GPNMB promoter was significantly associated with abdominal fat weight (AFW, P < 0.05) and abdominal fat percentage (AFP, P < 0.01). Express profile analysis of the GPNMB indicated that the gene was mainly expressed in abdominal fat tissue, and its expression level was strongly positively correlated with AFW (R2 = 0.6356, P = 4.10E−05) and AFP (R2 = 0.6450, P = 2.90E−05). We then investigated biological function of GPNMB on adipogenesis in chicken, and found that GPNMB could inhibit abdominal preadipocyte proliferation, but promote abdominal preadipocyte differentiation and lipid deposition. Furthermore, we explored regulatory mechanism of GPNMB gene in chicken, and detected one nonclassical estrogen regulatory element (AP1) and one peroxisome proliferator-activated receptor α (PPARα) responsive element in the 2 kb promoter region of GPNMB gene, and demonstrated that estrogen could up-regulate GPNMB mRNA expression in adipose tissue and primary abdominal preadipocytes, while PPARα could down-regulate GPNMB expression in primary preadipocytes. Taken together, this study brings new insights into understanding the function and transcriptional control of GPNMB gene, and provides genetic markers for breeding selection to improve abdominal fat traits in chicken.

As an essential genetic and economic trait, chicken feather color has long been an important research topic. To further understand the mechanism of melanin deposition associated with coloration in chicken feathers, we selected feather follicle tissues from the neck and wings of chickens with differently colored feathers (yellow, sub-Columbian, and silver) for transcriptome analysis. We focused on genes that were expressed in both the wings and neck and were expressed with the same trends in breeds with two different plumage colors, specifically, SLC45A2, GPNMB, MLPH, TYR, KIT, WNT11, and FZD1. GO and KEGG enrichment analyses showed the DEGs were enriched in melanin-related pathways, such as tyrosine metabolic pathway and melanogenesis, and PPI analysis highlighted the genes SLC45A2 and GPNMB as associated with melanin deposition. Verification experiments in chicken melanocytes demonstrated that these two genes promote melanocyte melanin deposition. These data enrich our knowledge of the mechanisms that regulate chicken feather color.

Skeletal muscle is an essential tissue in meat-producing animals, and meat-producing traits have been a hot topic in chicken genetic breeding research. Current research shows that creatine kinase M-type-like (CKM) is one of the most abundant proteins in skeletal muscle and plays an important role in the growth and development of skeletal muscle, but its role in the development of chicken skeletal muscle is still unclear. Via RNA sequencing (RNA-seq), we found that CKM was highly expressed in chicken breast muscle tissue. In this study, the expression profile of CKM was examined by quantitative real-time PCR (qPCR), and overexpression and RNA interference techniques were used to explore the functions of CKM in the proliferation, apoptosis and differentiation of chicken primary myoblasts (CPMs). It was shown that CKM was specifically highly expressed in breast muscle and leg muscle and was highly expressed in stage 16 embryonic muscle, while CKM inhibited proliferation, promoted the apoptosis and differentiation of CPMs and was involved in regulating chicken myogenesis. Transcriptome sequencing was used to identify genes that were differentially expressed in CPMs after CKM disruption, and bioinformatics analysis showed that CKM was involved in regulating chicken myogenesis. In summary, CKM plays an important role in skeletal muscle development during chicken growth and development.

Abstract Background Estrogen plays an essential role in female development and reproductive function. In chickens, estrogen is critical for lipid metabolism in the liver. The regulatory molecular network of estrogen in chicken liver is poorly understood. To identify estrogen-responsive genes and estrogen functional sites on a genome-wide scale, we determined expression profiles of mRNAs, lncRNAs, and miRNAs in estrogen-treated ((17β-estradiol)) and control chicken livers using RNA-Sequencing (RNA-Seq) and studied the estrogen receptor α binding sites by ChIP-Sequencing (ChIP-Seq). Results We identified a total of 990 estrogen-responsive genes, including 962 protein-coding genes, 11 miRNAs, and 17 lncRNAs. Functional enrichment analyses showed that the estrogen-responsive genes were highly enriched in lipid metabolism and biological processes. Integrated analysis of the data of RNA-Seq and ChIP-Seq, identified 191 genes directly targeted by estrogen, including 185 protein-coding genes, 4 miRNAs, and 2 lncRNAs. In vivo and in vitro experiments showed that estrogen decreased the mRNA expression of PPARGC1B, which had been reported to be linked with lipid metabolism, by directly increasing the expression of miR-144-3p. Conclusions These results increase our understanding of the functional network of estrogen in chicken liver and also reveal aspects of the molecular mechanism of estrogen-related lipid metabolism.

The effective protection and utilization of poultry resources depend on an accurate understanding of the genetic diversity and population structure. The breeding of the specialized poultry lineage “Yufen 1”, with its defined characteristics, was approved by the China Poultry Genetic Resource Committee in 2015. Thus, to investigate the relationship between the progenitor H line and other poultry breeds, the genetic diversity and population structure of “Yufen 1” H line (YF) were investigated and compared with those of 2 commercial chicken breeds, the ancestor breed Red Jungle Fowls, and 11 Chinese indigenous chicken breeds based on a whole-genome resequencing approach using 8,112,424 SNPs. The genetic diversity of YF was low, and the rate of linkage disequilibrium decay was significantly slower than that of the other Chinese indigenous breeds. In addition, it was shown that the YF population was strongly selected during intensive breeding and that genetic resources have been seriously threatened, which highlights the need to establish a systematic conservation strategy as well as utilization techniques to maintain genetic diversity within YF. Moreover, a principal component analysis, a neighbor-joining tree analysis, a structure analysis, and genetic differentiation indices indicated that YF harbors a distinctive genetic resource with a unique genetic structure separate from that of Chinese indigenous breeds at the genome level. The findings provide a valuable resource and the theoretical basis for the further conservation and utilization of YF.

Abstract Background Chicken skeletal muscle is an important economic product. The late stages of chicken development constitute the main period that affects meat production. LncRNAs play important roles in controlling the epigenetic process of growth and development. However, studies on the role of lncRNAs in the late stages of chicken breast muscle development are still lacking. In this study, to investigate the expression characteristics of lncRNAs during chicken muscle development, 12 cDNA libraries were constructed from Gushi chicken breast muscle samples from 6-, 14-, 22-, and 30-week-old chickens. Results A total of 1252 new lncRNAs and 1376 annotated lncRNAs were identified. Furthermore, 53, 61, 50, 153, 117, and 78 DE-lncRNAs were found in the W14 vs. W6, W22 vs. W14, W22 vs. W6, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparison groups, respectively. After GO enrichment analysis of the DE-lncRNAs, several muscle development-related GO terms were found in the W22 vs. W14 comparison group. Moreover, it was found that the MAPK signaling pathway was one of the most frequently enriched pathways in the different comparison groups. In addition, 12 common target DE-miRNAs of DE-lncRNAs were found in different comparison groups, some of which were muscle-specific miRNAs, such as gga-miR-206, gga-miR-1a-3p, and miR-133a-3p. Interestingly, the precursors of four newly identified miRNAs were found to be homologous to lncRNAs. Additionally, we found some ceRNA networks associated with muscle development-related GO terms. For example, the ceRNA networks contained the DYNLL2 gene with 12 lncRNAs that targeted 2 miRNAs. We also constructed PPI networks, such as IGF-I-EGF and FZD6-WNT11. Conclusions This study revealed, for the first time, the dynamic changes in lncRNA expression in Gushi chicken breast muscle at different periods and revealed that the MAPK signaling pathway plays a vital role in muscle development. Furthermore, MEF2C and its target lncRNA may be involved in muscle regulation through the MAPK signaling pathway. This research provided valuable resources for elucidating posttranscriptional regulatory mechanisms to promote the development of chicken breast muscles after hatching.

Abstract Background Domesticated chickens have a wide variety of phenotypes, in contrast with their wild progenitors. Unlike other chicken breeds, Xichuan black-bone chickens have blue-shelled eggs, and black meat, beaks, skin, bones, and legs. The breeding history and the economically important traits of this breed have not yet been explored at the genomic level. We therefore used whole genome resequencing to analyze the breeding history of the Xichuan black-bone chickens and to identify genes responsible for its unique phenotype. Results Principal component and population structure analysis showed that Xichuan black-bone chicken is in a distinct clade apart from eight other breeds. Linkage disequilibrium analysis showed that the selection intensity of Xichuan black-bone chickens is higher than for other chicken breeds. The estimated time of divergence between the Xichuan black-bone chickens and other breeds is 2.89 ka years ago. Fst analysis identified a selective sweep that contains genes related to melanogenesis. This region is probably associated with the black skin of the Xichuan black-bone chickens and may be the product of long-term artificial selection. A combined analysis of genomic and transcriptomic data suggests that the candidate gene related to the black-bone trait, EDN3, might interact with the upstream ncRNA LOC101747896 to generate black skin color during melanogenesis. Conclusions These findings help explain the unique genetic and phenotypic characteristics of Xichuan black-bone chickens, and provide basic research data for studying melanin deposition in animals.

Abstract Background Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection on the intestinal microflora of chickens. Results A 29-week commercial layer flock positive for avian leukosis virus-J (ALV-J), Marek’s disease virus (MDV) and avian reticuloendotheliosis virus (REV) was selected, and fresh fecal samples were collected and examined for the composition of the gut microflora by Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The operational taxonomic units (OTUs) of the fecal microbiota differentiated the chickens infected with only ALV-J and those coinfected with ALV-J and MDV or REV from infection-negative chickens. The enrichment and diversity of cloacal microflora in chickens infected with ALV-J alone were slightly different from those in the infection-negative chickens. However, the diversity of cloacal microflora was significantly increased in chickens coinfected with both ALV-J and MDV or REV. Conclusions The intestinal microbiota was more strongly disturbed in chickens after coinfection with ALV-J and MDV or REV than after infection with ALV-J alone, and there may be underlying mechanisms by which the capacity for the stabilization of the intestinal flora was impaired due to viral infection and tumorigenesis.

Abstract Background The SH3RF2 gene is a protein-coding gene located in a quantitative trait locus associated with body weight, and its deletion has been shown to be positively associated with body weight in chickens. Results In the present study, CNV in the SH3RF2 gene was detected in 4079 individuals from 17 populations, including the “Gushi ×Anka” F2 resource population and populations of Chinese native chickens, commercial layers, and commercial broilers. The F2 resource population was then used to investigate the genetic effects of the chicken SH3RF2 gene. The results showed that the local chickens and commercial layers were all homozygous for the wild-type allele. Deletion mutation individuals were detected in all of the commercial broiler breeds except Hubbard broiler. A total of, 798 individuals in the F2 resource group were used to analyze the effects of genotype (DD/ID/II) on chicken production traits. The results showed that CNV was associated with 2-, 6-, 10-, and 12-week body weight (P = 0.026, 0.042, 0.021 and 0.039 respectively) and significantly associated with 8-week breast bone length (P = 0.045). The mutation was significantly associated with 8-week body weight (P = 0.007) and 4-week breast bone length (P = 0.010). CNV was significantly associated with evisceration weight, leg muscle weight, carcass weight, breast muscle weight and gizzard weight (P = 0.032, 0.033, 0.045, 0.004 and 0.000, respectively). Conclusions CNV of the SH3RF2 gene contributed to variation in the growth and weight gain of chickens.

There are five indigenous chicken breeds in Henan Province, China. These breeds have their own unique phenotypic characteristics in terms of morphology, behavior, skin and feather color, and productive performance, but their genetic basis is not well understood. Therefore, we analyzed the genetic structure, genomic diversity, and migration history of Henan indigenous chicken populations and the selection signals and genes responsible for Henan gamecock unique phenotypes using whole genome resequencing. The results indicate that Henan native chickens clustered most closely with the chicken populations in neighboring provinces. Compared to other breeds, Henan gamecock’s inbreeding and selection intensity were more stringent. TreeMix analysis revealed the gene flow from southern chicken breeds into the Zhengyang sanhuang chicken and from the Xichuan black-bone chicken into the Gushi chicken. Selective sweep analysis identified several genes and biological processes/pathways that were related to body size, head control, muscle development, reproduction, and aggression control. Additionally, we confirmed the association between genotypes of SNPs in the strong selective gene LCORL and body size and muscle development in the Gushi-Anka F2 resource population. These findings made it easier to understand the traits of the germplasm and the potential for using the Henan indigenous chicken.

Molecular breeding can accelerate the process of animal breeding and improve the breeding efficiency. To date, many Indel molecular markers have been identified in livestock and poultry, but how Indels affect economic traits is not well understood. For molecular breeding, it is crucial to reveal the mechanism of action of Indels and to provide more accurate information. The purpose of this study was to investigate how the 52/224-bp multiallelic Indels of the chicken QPCTL promoter area affect the daily weight gain of chickens and the potential regulatory mechanism of the QPCTL gene. The analysis was conducted by association analysis, qPCR, dual-fluorescence assay and Western blotting. The results showed that Indels in the QPCTL promoter region were significantly associated with the daily weight gain in chickens and that QPCTL expression showed a decreasing trend in embryonic breast muscle tissues. Furthermore, QPCTL expression was significantly higher in breast muscle tissues of the AC genotype than in those of the AB and BB genotypes. Based on the transcriptional activity results, the pGL3-C vector produced more luciferase activity than pGL3-A and pGL3-B. In addition, overexpression of QPCTL promoted chicken primary myoblast (CPM) proliferation and inhibited differentiation. The results of this study suggest that Indels in the promoter region of the QPCTL gene may regulate the proliferation and differentiation of CPMs by affecting the expression of QPCTL, which ultimately affects the growth rate of chickens. These Indels have important value for the molecular breeding of chickens, and QPCTL can be used as a candidate gene to regulate and improve chicken growth and development.

Skeletal muscle development and intramuscular fat (IMF) content, which positively contribute to meat production and quality, are regulated by precisely orchestrated processes. However, changes in three-dimensional chromatin structure and interaction, a newly emerged mediator of gene expression, during the skeletal muscle development and IMF deposition have remained unclear. In the present study, we analyzed the differences in muscle development and IMF content between one-day-old commercial Arbor Acres broiler (AA) and Chinese indigenous Lushi blue-shelled-egg chicken (LS) and performed Hi-C analysis on their breast muscles. Our results indicated that significantly higher IMF content, however remarkably lower muscle fiber diameter was detected in breast muscle of LS chicken compared to that of AA broiler. The chromatin intra-interaction was prior to inter-interaction in both AA and LS chicken, and chromatin inter-interaction was heavily focused on the small and gene-rich chromosomes. For genomic compartmentalization, no significant difference in the number of B type compartments was found, but AA had more A type compartments versus LS. The A/B compartment switching of AA versus LS showed more A to B switching than B to A switching. There were no significant differences in the average sizes and distributions of topologically associating domains (TAD). Additionally, approximately 50% of TAD boundaries were overlapping. The reforming and disappearing events of TAD boundaries were identified between AA and LS chicken breast muscles. Among these, the HMGCR gene was located in the TAD-boundary regions in AA broilers, but in TAD-interior regions in LS chickens, and the IGF2BP3 gene was located in the AA-unique TAD boundaries. Both HMGCR and IGF2BP3 genes exhibited increased mRNA expression in one-day-old AA broiler breast muscles. It was demonstrated that the IGF2BP3 and HMGCR genes regulated by TAD boundary sliding were potential biomarkers for chicken breast muscle development and IMF deposition. Our data not only provide a valuable understanding of higher-order chromatin dynamics during muscle development and lipid accumulation but also reveal new insights into the regulatory mechanisms of muscle development and IMF deposition in chicken.

Along with sexual maturity, the liver undergoes numerous metabolic processes to adapt the physiological changes associated with egg-laying in hens. However, mechanisms regulating the processes were unclear. In this study, comparative hepatic proteome and acetyl-proteome between pre- and peak-laying hens were performed. The results showed that the upregulated proteins were mainly related to lipid and protein biosynthesis, while the downregulated proteins were mainly involved in pyruvate metabolism and were capable of inhibiting gluconeogenesis and lactate synthesis in peak-laying hens compared with that in pre-laying hens. With unchanged expression level, the significant acetylated proteins were largely functioned on activation of polyunsaturated fatty acid oxidation in peroxisome, while the significant deacetylated proteins were principally used to elevate medium and short fatty acid oxidation in mitochondria and oxidative phosphorylation. Most of the proteins which involved in gluconeogenesis, lipid transport, and detoxification were influenced by both protein expression and acetylation. Taken overall, a novel mechanism wherein an alternate source of acetyl coenzyme A was produced by activation of FA oxidation and pyruvate metabolism to meet the increased energy demand and lipid synthesis in liver of laying hens was uncovered. This study provides new insights into molecular mechanism of adaptation to physiological changes in liver of laying hens.

Analyzing marker-assisted breeding is an important method utilized in modern molecular breeding. Recent studies have determined that a large number of molecular markers appear to explain the impact of “lost heritability” on human height. Therefore, it is necessary to locate molecular marker sites in poultry and investigate the possible molecular mechanisms governing their effects. In this study, we found a 104-bp insertion/deletion polymorphism in the 5′UTR of the ADPRHL1 gene through resequencing. In cross-designed F2 resource groups, the indel was significantly associated with weight at 0, 2, 4, 6, and 10 weeks and a number of other traits [carcass weight (CW), semi-evisceration weight (SEW), evisceration weight (EW), claw weight (CLW), wings weight (DWW), gizzard weight (GW), pancreas weight (PW), chest muscle weight (CMW), leg weight (LW), leg muscle weight (LMW), shedding Weight (SW), liver rate (LR), and leg muscle rate (LMR)] (P < 0.05). In brief, the insertion-insertion (II) genotype was significantly associated with the greatest growth traits and meat quality traits, whereas the values associated with the insertion-deletion (ID) genotype were the lowest in the F2 reciprocal cross chickens. The mutation sites were genotyped in 4,526 individuals from 12 different chicken breeds and cross-designed F2 resource groups. The II genotype is the most important genotype in commercial broilers, and the I allele frequency observed in these breeds is relatively high. Deletion mutations tend to be fixed in commercial broilers. However, there is still considerable great potential for breeding in dual-purpose chickens and commercial laying hens. A luciferase reporter assay showed that the II genotype of the ADPRHL1 gene possessed 2.49-fold higher promoter activity than the DD genotype (P < 0.05). We hypothesized that this indel might affect the transcriptional activity of ADPRHL1, thereby affecting the growth traits of chickens. These findings may help to elucidate the function of the ADPRHL1 gene and facilitate enhanced reproduction in the chicken industry.

Abstract Background The molecular mechanisms underlying stress-influenced immune function of chicken (Gallus Gallus) are not clear. The stress models can be established effectively by feeding chickens corticosterone (CORT) hormone. The bursa of Fabricius is a unique central immune organ of birds. RNA-Seq technology was used to investigate differences in the expression profiles of immune-related genes and associated pathways in the bursa of Fabricius to clarify molecular mechanisms. The aim of this study was to broaden the understanding of the stress-influenced immune function in chickens. Results Differentially expressed genes (DEGs) in the bursa of Fabricius between experimental group (basal diet with added CORT 30 mg/kg; C_B group) and control group (basal diet; B_B group) were identified by using RNA-seq technology. In total, we found 1434 significant DEGs (SDEGs), which included 199 upregulated and 1235 downregulated genes in the C_B group compared with the B_B group. The immune system process GO term was the top significantly GO term, including MYD88, TLR4, IL15, VEGFA gene and so on. The cytokine-cytokine receptor interaction pathway and the Toll-like receptor signaling pathway were the key pathways affected by stress. The protein-protein interaction (PPI) analysis of the SDEGs showed that VEGFA, MyD88 and IL15 were hub genes and module analysis showed that MYD88, TLR4 and VEGFA play important roles in response to stress. Conclusion This study showed that the VEGFA and ILs (such as IL15) via the cytokine-cytokine receptor interaction pathway, MYD88 and TLR4 via the Toll-like receptor signaling pathway may play important roles in the regulation of immune function under stress condition with CORT administration. The results of this study provide a reference for further studies of the molecular mechanisms of stress-influenced immune function.

Intramuscular fat (IMF), which regulated by genetics, nutrition and environment is an important factor that influencing meat quality. Up to now, the epigenetic regulation mechanism underlying poultry IMF deposition remains poorly understood. Here, we focused on the DNA methylation, which usually regulate genes in transcription level. To look into the essential role of DNA methylation on the IMF deposition, chicken intramuscular preadipocytes were isolated and cultured in vitro, and a model of intramuscular adipocyte differentiation was constructed. Combined the whole genome bisulfite sequencing (WGBS) and RNA-Seq technologies, we identified several methylated genes, which mainly affecting fatty acid metabolism and muscle development. Furthermore, we reported that DNA methylation regulate intramuscular adipogenesis by regulating the genes, such as collagen, type VI, alpha 1 (COL6A1) thus affecting IMF deposition. Overexpression of COL6A1 increases the lipid droplet and inhibits cell proliferation by regulating CHAD and CAMK2 in intramuscular adipocytes, while knockdown of COL6A1 shows the opposite effect. Taken together, our results reveal that DNA methylation plays an important role in poultry IMF deposition.

Liver is the main organ of lipid metabolism in chicken, especially for laying hens. To explore the molecular mechanism of lipid metabolism in chicken, five novel genes discovered in chicken liver tissue were systematically studied. Bioinformatic analysis was used to analyze the gene characteristics. The expression patterns and regulatory molecular mechanism of the five genes were examined. Our results showed that all five novel genes contain a common NADP-binding site that belongs to the NADB-Rossmann superfamily, and the genes were designated NADB-LER1-5. Phylogenetic tree of the NADB-LERs gene family in different species suggested these five genes originated from the same ancestor. Tissue distributions showed that NADB-LER1-4 genes were highly expressed in lipid metabolism organs, including liver, kidney and duodenum, and that the NADB-LER5 gene was highly expressed in liver and kidney. The spatiotemporal expression indicated that the expression levels of NADB-LER1-5 genes in liver tissue were significantly greater in sexually mature hens than that of immature pullets (P-value ≤ 0.05). The expression levels of NADB-LER1-5 were significantly induced by 17β-estradiol in primary cultured chicken embryo hepatocytes (P-value ≤ 0.05), and 17β-estradiol regulated the expression of NADB-LER1-5 mediated by ERα. Individual assays verified that under induction of 17β-estradiol, the five novel genes were significantly upregulated, with subsequent alteration in serum TG, TC, and VLDLs in 10-week-old pullets. This study proved NADB-LERs family mainly expressed in liver, kidney, and duodenum tissues. 17β-estradiol induces the expression of NADB-LER1-5 genes predominantly mediated via ERα. They likely involved in lipid metabolism in the liver of chicken.

BackgroundBone abnormality and leg disease in commercial broiler flocks are increasingly prominent, causing serious economic losses to the broiler breeding industry. Valgus-varus deformity (VVD) is a common deformity of the long bone in broilers that manifests as an outward or inward deviation of the tibiotarsus or tarsometatarsus. There is a paucity of studies on the molecular mechanisms of VVD.ResultsIn this study, 6 cDNA libraries were constructed from spleen samples from VVD birds and normal birds. A total of 1951 annotated lncRNAs, 7943 novel lncRNAs and 30252 mRNAs were identified by RNA-sequencing. In addition, 420 differentially expressed (DE) mRNAs and 124 differentially expressed lncRNAs (adjusted P-value < 0.05) were obtained. A total of 16 dysregulated genes were confirmed by qPCR to be consistent with the results of the RNA-Seq. The functional lncRNA-mRNA co-expression network was constructed using differentially expressed mRNAs and target genes of the differentially expressed lncRNAs. 11 DE genes were obtained from the analysis. In order to gain insight into the interactions of genes, lncRNAs and pathways associated with VVD, we focused on the following pathways, which are involved in immunity and bone development: the Jak-stat signaling pathway, Toll-like receptor signaling pathway, Wnt-signaling pathway, mTOR signaling pathway, VEGF signaling pathway, Notch signaling pathway, TGF-beta signaling pathway and Fanconi anemia pathway. All together, 30 candidate DE genes were obtained from these pathways. We then analyzed the interaction between the DE genes and their corresponding lncRNAs. From these interaction network analyses we found that GARS, NFIC, PIK3R1, BMP6, NOTCH1, ACTB and CREBBP were the key core nodes of these networks.ConclusionThis study showed that differentially expressed genes and signaling pathways were related to immunity or bone development. These results increase the understanding of the molecular mechanisms of VVD and provide some reference for the etiology and pathogenesis of VVD.

Abstract Background The intracerebroventricular injection of visfatin increases feed intake. However, little is known about the molecular mechanism in chicks. This study was conducted to assess the effect of visfatin on the feeding behavior of chicks and the associated molecular mechanism. Results In response to the intraventricular injection of 40 ng and 400 ng visfatin, feed intake in chicks was significantly increased, and the concentrations of glucose, insulin, TG, HDL and LDL were significantly altered. Using RNA-seq, we identified DEGs in the chick hypothalamus at 60 min after injection with various doses of visfatin. In total, 325, 85 and 519 DEGs were identified in the treated chick hypothalamus in the LT vs C, HT vs C and LT vs HT comparisons, respectively. The changes in the expression profiles of DEGs, GO functional categories, KEGG pathways, and PPI networks by visfatin-mediated regulation of feed intake were analyzed. The DEGs were grouped into 8 clusters based on their expression patterns via K-mean clustering; there were 14 appetite-related DEGs enriched in the hormone activity GO term. The neuroactive ligand-receptor interaction pathway was the key pathway affected by visfatin. The PPI analysis of DEGs showed that POMC was a hub gene that interacted with the maximum number of nodes and ingestion-related pathways, including POMC, CRH, AgRP, NPY, TRH, VIP, NPYL, CGA and TSHB. Conclusion These common DEGs were enriched in the hormone activity GO term and the neuroactive ligand-receptor interaction pathway. Therefore, visfatin causes hyperphagia via the POMC/CRH and NPY/AgRP signaling pathways. These results provide valuable information about the molecular mechanisms of the regulation of food intake by visfatin.

The Xichuan black-bone chicken, which is a rare local chicken species in China, is an important genetic resource of black-bone chickens. Tyrosine can affect melanin production, but the molecular mechanism underlying tyrosine-induced melanin deposition in Xichuan black-bone chickens is poorly understood. Here, the blackness degree and melanin content of the breast muscle of Xichuan black-bone chickens fed a basic diet with five levels of added tyrosine (i.e., 0.2, 0.4, 0.6, 0.8, and 1.0%; these groups were denoted test groups I-V, respectively) were assessed, and the results showed that 0.8% tyrosine was the optimal level of added tyrosine. Moreover, the effects of tyrosine supplementation on the proliferation and tyrosinase content of melanocytes in Xichuan black-bone chickens were evaluated. The results revealed a dose-dependent relationship between tyrosine supplementation and melanocyte proliferation. In addition, 417 differentially expressed genes (DEGs), including 160 upregulated genes and 257 downregulated genes, were identified in a comparative analysis of the transcriptome profiles constructed using the pooled total RNA from breast muscle tissues of the control group and test group IV, respectively (fold change ≥2.0, P < 0.05). These DEGs were mainly involved in melanogenesis, the calcium signaling pathway, the Wnt signaling pathway, the mTOR signaling pathway, and vascular smooth muscle contraction. The pathway analysis of the DEGs identified some key genes associated with pigmentation, such as DCT and EDNRB2. In summary, the melanin content of breast muscle could be markedly enhanced by adding an appropriate amount of tyrosine to the diet of Xichuan black-bone chickens, and the EDNRB2-mediated molecular regulatory network could play a key role in the biological process of tyrosine-induced melanin deposition. These results have deepened the understanding of the molecular regulatory mechanism of melanin deposition in black-bone chickens and provide a basis for the regulation of nutrition and genetic breeding associated with melanin deposition in Xichuan black-bone chickens.

There is a lack of understanding surrounding the molecular mechanisms involved in the development of chicken skeletal muscle in the late postnatal stage, especially in the regulation of breast muscle development related genes, pathways, miRNAs and other factors. In this study, 12 cDNA libraries and 4 small RNA libraries were constructed from Gushi chicken breast muscle samples from 6, 14, 22, and 30 weeks. A total of 15,508 known transcripts, 25,718 novel transcripts, 388 known miRNAs and 31 novel miRNAs were identified by RNA-seq in breast muscle at the four developmental stages. Through correlation analysis of miRNA and mRNA expression profiles, it was found that 417, 370, 240, 1,418, 496, and 363 negatively correlated miRNA–mRNA pairs of W14 vs. W6, W22 vs. W6, W22 vs. W14, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparisons, respectively. Based on the annotation analysis of these miRNA–mRNA pairs, we constructed the miRNA–mRNA interaction network related to biological processes, such as muscle cell differentiation, striated muscle tissue development and skeletal muscle cell differentiation. The interaction networks for signaling pathways related to five KEGG pathways (the focal adhesion, ECM-receptor interaction, FoxO signaling, cell cycle, and p53 signaling pathways) and PPI networks were also constructed. We found that ANKRD1, EYA2, JSC, AGT, MYBPC3, MYH11, ACTC1, FHL2, RCAN1, FOS, EGR1, and FOXO3, PTEN, AKT1, GADD45, PLK1, CCNB2, CCNB3 and other genes were the key core nodes of these networks, most of which are targets of miRNAs. The FoxO signaling pathway was in the center of the five pathway-related networks. In the PPI network, there was a clear interaction among PLK1 and CDK1, CCNB2, CDK1, and GADD45B, and CDC45, ORC1 and MCM3 genes. These results increase the understanding for the molecular mechanisms of chicken breast muscle development, and also provide a basis for studying the interactions between genes and miRNAs, as well as the functions of the pathways involved in postnatal developmental regulation of chicken breast muscle.

The formation mechanism underlying the blue eggshell characteristic has been discovered in birds, and SLCO1B3 is the key gene that regulates the blue eggshell color. Insertion of an endogenous retrovirus, EAV-HP, in the SLCO1B3 5′ flanking region promotes SLCO1B3 expression in the chicken shell gland, and this expression causes bile salts to enter the shell gland, where biliverdin is secreted into the eggshell, forming a blue shell. However, at different laying stages of the same group of chickens, the color of the eggshell can vary widely, and the molecular mechanism underlying the eggshell color change remains unknown. Therefore, to reveal the molecular mechanism of the blue eggshell color variations, we analyzed the change in the eggshell color during the laying period. The results indicated that the eggshell color in Lushi chickens can be divided into three stages: 20–25 weeks for dark blue, 26–45 weeks for medium blue, and 46–60 weeks for light blue. We further investigated the expression and methylation levels of the SLCO1B3 gene at eight different weeks, finding that the relative expression of SLCO1B3 was significantly higher at 25 and 30 weeks than at other laying weeks. Furthermore, the overall methylation rate of the SLCO1B3 gene in Lushi chickens increased gradually with increasing weeks of egg production, as shown by bisulfite sequencing PCR. Pearson correlation analysis showed that methylation of the promoter region of SLCO1B3 was significantly negatively correlated with both SLCO1B3 expression in the shell gland tissue and eggshell color. In addition, we predicted that CpG5 and CpG8 may be key sites for regulating SLCO1B3 gene transcription. Our findings show that as the level of methylation increases, methylation of the CpG5 and CpG8 sites hinders the binding of transcription factors to the promoter, reducing SLCO1B3 expression during the late period and resulting in a lighter eggshell color.

Long noncoding RNAs (lncRNAs) play important roles in transcriptional and posttranscriptional regulation. However, the effects of lncRNAs on the meat quality of chicken hasn't been elucidated clearly yet. Gushi chickens are popular in China because of their superior meat quality, particularly the tender flesh, and unique flavor. Gushi chickens are popular in China because of their superior meat quality, delicate flesh, and unique flavor. We performed RNA-Seq analysis of breast muscle from Gushi chicken at two physiological stages, including juvenile (G20W) and laying (G55W). In total, 186 lncRNAs and 881 mRNAs were differentially expressed between G20W and G55W (fold change ≥ 2.0, P < 0.05). Among them, 131 lncRNAs presented upregulated and 55 were downregulated. We identified the cis and trans target genes of the differentially expressed lncRNAs, and constructed lncRNA-mRNA interaction networks. The results showed that differentially expressed mRNAs and lncRNAs were mainly involved in ECM-receptor interaction, glycerophospholipid metabolism, ubiquitin-mediated proteolysis, and the biosynthesis of amino acids. In summary, our study utilized RNA-seq analysis to predict the functions of lncRNA on chicken meat quality. Furthermore, comprehensive analysis identified lncRNAs and their target genes, which may contribute to a better understanding of the molecular mechanisms underlying in poultry meat quality and provide a theoretical basis for further research.

BACKGROUND:Coloration is one of the most recognizable characteristics in chickens, and clarifying the coloration mechanisms will help us understand feather color formation. "Yufen I" is a commercial egg-laying chicken breed in China that was developed by a three-line cross using lines H, N and D. Columbian plumage is a typical feather character of the "Yufen I" H line. To elucidate the molecular mechanism underlying the pigmentation of Columbian plumage, this study utilizes high-throughput sequencing technology to compare the transcriptome and proteome differences in the follicular tissue of different feathers, including the dorsal neck with black and white striped feather follicles (Group A) and the ventral neck with white feather follicles (Group B) in the "Yufen I" H line. RESULTS:In this study, we identified a total of 21,306 genes and 5,203 proteins in chicken feather follicles. Among these, 209 genes and 382 proteins were differentially expressed in two locations, Group A and Group B, respectively. A total of 8 differentially expressed genes (DEGs) and 9 differentially expressed proteins (DEPs) were found to be involved in the melanogenesis pathway. Additionally, a specifically expressed MED23 gene and a differentially expressed GNAQ protein were involved in melanin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapped 190 DEGs and 322 DEPs to 175 and 242 pathways, respectively, and there were 166 pathways correlated with both DEGs and DEPs. 49 DEPs/DEGs overlapped and were enriched for 12 pathways. Transcriptomic and proteomic analyses revealed that the following pathways were activated: melanogenesis, cardiomyocyte adrenergic, calcium and cGMP-PKG. The expression of DEGs was validated by real-time quantitative polymerase chain reaction (qRT-PCR) that produced results similar to those from RNA-seq. In addition, we found that the expression of the MED23, FZD10, WNT7B and WNT11 genes peaked at approximately 8 weeks in the "Yufen I" H line, which is consistent with the molting cycle. As both groups showed significant differences in terms of the expression of the studied genes, this work opens up avenues for research in the future to assess their exact function in determining plumage color. CONCLUSION:Common DEGs and DEPs were enriched in the melanogenesis pathway. MED23 and GNAQ were also reported to play a crucial role in melanin synthesis. In addition, this study is the first to reveal gene and protein variations in in the "Yufen I" H line during Columbian feather color development and to discover principal genes and proteins that will aid in functional genomics studies in the future. The results of the present study provide a significant conceptual basis for the future breeding schemes with the "Yufen I" H line and provide a basis for research on the mechanisms of feather pigmentation.

The excessive deposition of abdominal fat has become an important factor in restricting the production efficiency of chickens, so reducing abdominal fat deposition is important for improving growth rate. It has been proven that miRNAs play an important role in regulating many physiological processes of organisms. In this study, we constructed a model of adipogenesis by isolating preadipocytes (Ab-Pre) derived from abdominal adipose tissue and differentiated adipocytes (Ab-Ad) in vitro. Deep sequencing of miRNAs and mRNAs expressed in Ab-Pre and Ab-Ad groups was conducted to explore the effect of miRNAs and mRNAs on fat deposition. We identified 80 differentially expressed miRNAs (DEMs) candidates, 58 of which were up-regulated and 22 down-regulated. Furthermore, six miRNAs and six mRNAs were verified by qRT-PCR, and the results showed that the expression of the DEMs and differentially expressed genes (DEGs) in the two groups was consistent with our sequencing results. When target genes of miRNA were combined with mRNA transcriptome data, a total of 891 intersection genes were obtained, we predicted the signal pathways of cross genes enrichment to the MAPK signal pathway, insulin signal pathway, fatty acid metabolism, and ECM−receptor interaction. Meanwhile, we constructed miRNA and negatively correlated mRNA target networks, including 12 miRNA−mRNAs pairs, which showed a strong association with the abdominal adipocyte differentiation (miR-214−ACSBG2, NFKB2, CAMK2A, ACLY, CCND3, PLK3, ITGB2; miR-148a-5p−ROCK2; miR-10a-5p−ELOVL5; miR-146b-5p−LAMA4; miR-6615-5p−FLNB; miR-1774−COL6A1). Overall, these findings provide a background for further research on lipid metabolism. Thus, we can better understand the molecular genetic mechanism of chicken abdominal fat deposition.

Leptin receptor (LEPR) belongs to the class I cytokine receptor superfamily which share common structural features and signal transduction pathways. Although multiple LEPR isoforms, which are derived from one gene, were identified in mammals, they were rarely found in avian except the long LEPR. Four alternative splicing variants of quail LEPR (qLEPR) had been cloned and sequenced for the first time (Wang et al., 2015 [1]). To define patterns of the four splicing variants (qLEPRl, qLEPR-a, qLEPR-b and qLEPR-c) and locate the conserved regions of qLEPRl, this data article provides nucleotide sequence alignment of qLEPR and amino acid sequence alignment of representative vertebrate LEPR. The detailed analysis was shown in [1]. Keywords: Quail LEPR, Alternative splicing, Evolutionary conservation, Sequence alignment

The molecular mechanisms underlying meat quality and muscle growth are not clear. The meat quality and growth rates of local chickens and commercial broilers are very different. The Ribo-Zero RNA-Seq technology is an effective means of analyzing transcript groups to clarify molecular mechanisms. The aim of this study was to provide a reference for studies of the differences in the meat quality and growth of different breeds of chickens.Ribo-Zero RNA-Seq technology was used to analyze the pectoral muscle transcriptomes of Gushi chickens and AA broilers. Compared with AA broilers, 1649 genes with annotated information were significantly differentially expressed (736 upregulated and 913 downregulated) in Gushi chickens with Q≤0.05 (Q is the P-value corrected by multiple assumptions test) at a fold change ≥2 or ≤0.5. In addition, 2540 novel significantly differentially expressed (SDE) genes (1405 upregulated and 1135 downregulated) were discovered. The results showed that the main signal transduction pathways that differed between Gushi chickens and AA broilers were related to amino acid metabolism. Amino acids are important for protein synthesis, and they regulate key metabolic pathways to improve the growth, development and reproduction of organisms.This study showed that differentially expressed genes in the pectoral tissues of Gushi chickens and AA broilers were related to fat metabolism, which affects meat. Additionally, a large number of novel genes were found that may be involved in fat metabolism and thus may affect the formation of meat, which requires further study. The results of this study provide a reference for further studies of the molecular mechanisms of meat formation.

Intramuscular fat (IMF) is the most important evaluating indicator of chicken meat quality, the content of which is positively correlated with tenderness, flavor, and succulence of the meat. Chicken IMF deposition process is regulated by many factors, including genetic, nutrition, and environment. Although large number of omics’ studies focused on the IMF deposition process, the molecular mechanism of chicken IMF deposition is still poorly understood. In order to study the role of miRNAs in chicken intramuscular adipogenesis, the intramuscular adipocyte differentiation model (IMF-preadipocytes and IMF-adipocytes) was established and subject to miRNA-Seq. A total of 117 differentially expressed miRNAs between two groups were obtained. Target genes prediction and functional enrichment analysis revealed that eight pathways involved in lipid metabolism related processes, such as fatty acid metabolism and fatty acid elongation. Meanwhile a putative miRNA, gga-miR-18b-3p, was identified be served a function in the intramuscular adipocyte differentiation. Luciferase assay suggested that the gga-miR-18b-3p targeted to the 3′UTR of ACOT13. Subsequent functional experiments demonstrated that gga-miR-18b-3p acted as an inhibitor of intramuscular adipocyte differentiation by targeting ACOT13. Our findings laid a new theoretical foundation for the study of lipid metabolism, and also provided a potential target to improve the meat quality in the poultry industry.