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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., (© 2024. The Author(s).)

Long noncoding RNAs (lncRNAs) participate in regulating skeletal muscle development. However, little is known about their role in regulating chicken myogenesis. In this study, we identified a novel lncRNA, lncMPD2, through transcriptome sequencing of chicken myoblasts at different developmental stages. Functionally, gain- and loss-of-function experiments showed that lncMPD2 inhibited myoblast proliferation and differentiation. Mechanistically, lncMPD2 directly bound to miR-34a-5p, and miR-34a-5p promoted myoblasts proliferation and differentiation and inhibited the mRNA and protein expression of its target gene THBS1. THBS1 inhibited myoblast proliferation and differentiation in vitro and delayed muscle regeneration in vivo. Furthermore, rescue experiments showed that lncMPD2 counteracted the inhibitory effects of miR-34a-5p on THBS1 and myogenesis-related gene mRNA and protein expression. In conclusion, lncMPD2 regulates the miR-34a-5p/THBS1 axis to inhibit the proliferation and differentiation of myoblasts and skeletal muscle regeneration. This study provides more insight into the molecular regulatory network of skeletal muscle development, identifying novel potential biomarkers for improving chicken quality and increasing chicken yield. In addition, this study provides a potential goal for breeding strategies that minimize muscle damage in chickens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Valgus-varus deformity (VVD) is a common long bone deformity in broilers. Imbalance in cartilage homeostasis is the main feature of leg disease. Exosomes act as an important intercellular communication vector that regulates chondrogenesis by encapsulating specific nucleic acids and proteins. However, the exact mechanism of how plasma exosomal miRNAs regulate cartilage homeostasis in VVD broilers remains unclear. This study first demonstrated the structural disorder, growth retardation, and reduced proliferative capacity of VVD cartilage in vitro and in vivo. Subsequently, VVD and Normal broiler plasma exosomes were collected for miRNA sequencing. Cartilage-specific miR-455-5p was extraordinarily emphasized by performing bioinformatics analysis on differential miRNA target genes and further validated by tissue expression profiling. PKH67 fluorescently labeled plasma exosomes were shown to be taken up by chondrocytes, deliver miR-455-5p, inhibit chondrocyte proliferation, and disrupt their homeostasis, and these effects could be inhibited by the miR-inhibitors. Mechanistically, MiR-455-5p targets Ribosomal Protein S6 Kinase B1 (RPS6KB1) to inhibit RPS6 phosphorylation and reduce the synthesis of key proteins for cartilage proliferation, which in turn inhibits cartilage proliferation and disrupts its homeostasis. In conclusion, the present study identified abnormalities in VVD cartilage tissue and clarified the specific mechanism by which plasma exosome-derived miR-455-5p regulates cartilage homeostasis., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

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., (© 2024. The Author(s).)

LIM domain binding 3 ( LDB3 ) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants ( LDB3 -X, LDB3 -XN1, and LDB3 -XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3 -XN1 and LDB3 -XN2 are novel variants. LDB3 -X and LDB3 -XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3 -XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20 . The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC , which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein-protein interactions.

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., (© 2024. The Author(s).)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (-log 10 (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., (© 2023. The Author(s).)

Alkaline phosphatase (ALP) is a marker of osteoblast maturation and an important indicator of bone metabolism. The activity of ALP can reflect the bone metabolism and growth traits of animals, so the polymorphism affecting ALP expression deserves further study. In this study, we identified an SNP site in ST3GAL4 found by genome-wide association studies (GWAS) in previous studies, 8 SNPs were also identified by DNA sequencing. Interestingly, there were 4 SNPs (rs475471G > A, rs475533C > T, rs475621A > G, rs475647C > A) completely linked by haplotype analysis. Therefore, we selected a tag SNP rs475471G > A to further analyze the ALP level of different genotypes in Hubbard leg disease population and an F 2 chicken resource population produced by Anka and Gushi chickens and carried out population genetic analysis in 18 chicken breeds. Association analysis showed that this QTL within ST3GAL4 was highly correlated with ALP level. The mutant individuals with genotype AA had the highest ALP level, followed by GA and GG carriers. The mutant individual carriers of AA and GA genotype had higher values for body weight (BW), chest width (CW), body slanting length (BSL), pelvis width (PW) at 4-week, the semi-evisceration weight (SEW), evisceration weight (EW) and Leg weight (LW) than GG genotypes. The amplification and typing of 4852 DNA samples from 18 different breeds showed GG genotype mainly existed in egg-type chickens and dual-type chickens, while the AA genotype was mainly distributed in commercial broilers and F 2 resource population. The individual carriers of the AA genotype had the highest ALP and showed better growth performance. Besides, tissue expression analysis used Cobb broiler showed significant differences between different genotypes in the spleen and duodenum. Taken together, this was the first time to determine 9 SNPs within ST3GAL4 related to ALP in chickens, 4 of them were complete linkage with each other, which provides useful information on the mutation of ST3GAL4 and could predict the serum ALP level of chicken early and as an effective potential molecular breeding marker for chickens., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

A growing number of studies have shown that members of the ankyrin repeat and suppressors of cytokine signaling (SOCS) box-containing protein (ASB) family are extensively involved in biological processes such as cell growth, tissue development, insulin signaling, ubiquitination, protein degradation, and skeletal muscle membrane protein formation, while the specific biological role of ankyrin-repeat and SOCS box protein 9 ( ASB9 ) remains unclear. In this study, a 21 bp indel in the intron of ASB9 was identified for the first time in 2641 individuals from 11 different breeds and an F 2 resource population, and differences were observed among individuals with different genotypes (II, ID, and DD). An association study of a cross-designed F 2 resource population revealed that the 21-bp indel was significantly related to growth and carcass traits. The significantly associated growth traits were body weight (BW) at 4, 6, 8, 10, and 12 weeks of age; sternal length (SL) at 4, 8, and 12 weeks of age; body slope length (BSL) at 4, 8, and 12 weeks of age; shank girth (SG) at 4 and 12 weeks of age; tibia length (TL) at 12 weeks of age; and pelvic width (PW) at 4 weeks of age ( p < 0.05). This indel was also significantly correlated with carcass traits including semievisceration weight (SEW), evisceration weight (EW), claw weight (CLW), breast muscle weight (BMW), leg weight (LeW), leg muscle weight (LMW), claw rate (CLR), and shedding weight (ShW) ( p < 0.05). In commercial broilers, the II genotype was the dominant genotype and underwent extensive selection. Interestingly, the ASB9 gene was expressed at significantly higher levels in the leg muscles of Arbor Acres broilers than those of Lushi chickens, while the opposite was true for the breast muscles. In summary, the 21-bp indel in the ASB9 gene significantly influenced the expression of the ASB9 gene in muscle tissue and was associated with multiple growth and carcass traits in the F 2 resource population. These findings suggested that the 21-bp indel within the ASB9 gene could be used in marker-assisted selection breeding for traits related to chicken growth.

In this study, the effects of Pseudostellaria heterophylla polysaccharide (PHP) on the growth, development, and liver metabolism of chicks were investigated by feeding chicks diets. Four hundred 7-d-old Gushi roosters were selected and randomly divided into four groups, labeled A, B, C, and D. Group A was fed the basal diet, and Groups B, C, and D were fed 100, 200, and 400 mg PHP per kilogram of basal diet, respectively. At 14, 21, 28 and 35 d of age, five chicks were randomly selected from each group to collect samples for index detection. The results showed that compared with Group A, there were significant reduction in average daily feed intake (ADFI) and feed-to-weight ratio (F/G) at 14, 21, and 28 d (P < 0.05), significant increase in average daily gain (ADG) at 21, 28 d (P < 0.05), significantly increased levels of total protein (TP), albumin (ALB), insulin (INS), thyroxine (T3), growth hormone (GH) at 14, 28 d (P < 0.05), significantly decreased levels of glucose (GLU), total cholesterol (TC), glucagon (GC), and triglyceride (TG) at 28 d in Group C (P < 0.05). There were significantly increased levels of TP, ALB at 14, 21 d (P < 0.05), significantly increased level of TP at 35 d (P < 0.05), significantly increased level of GH at 28 d (P < 0.05), significantly decreased levels of GLU, GC at 28 d (P < 0.05), significant reduction in F/G at 14, 21 d in Groups B and D (P < 0.05). Based on the above results, the livers from chicks in Groups A and C at 28 d were selected for transcriptome sequencing. The sequencing results showed that significantly differentially expressed genes (SDEGs) were enriched in growth and development, oxidative phosphorylation, the PPAR signaling pathway and the lipid metabolism pathway. All these results revealed that the addition of 200 mg/kg PHP in the diet promoted the growth and development, lipid metabolism and energy metabolism of chicks, inhibit inflammation and tumor development, and improve the function of the liver., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In order to investigate the regulatory role of the myeloid differentiation factor 88 (MyD88) gene in the stress inflammatory response to chicken spleen, the chicken stress model and macrophage (HD11) inflammation model were constructed in this study. Enzyme-linked immunosorbent assay and quantitative real-time PCR were used to investigate the effects of MyD88 on immune and inflammatory indicators. The results demonstrated that the levels of IgG, CD3+ and CD4+ in the serum of chickens in the beak trimming stress and heat stress groups decreased significantly compared to the control group without stress (P < 0.05), and the inflammation-related indices IL-1β, TNF-α, IL-6 and NF-κB increased significantly (P < 0.05). Stress up-regulated the expression levels of MyD88, IL-1β, NF-κB and TLR4 in the spleen, stimulated the release of inflammatory factors. Overexpression of MyD88 significantly up-regulated the expression levels of the inflammatory factors IL-1β, TNF-α, IL-8, NF-κB and TLR4 in HD11 cells (P < 0.05). Co-treatment with lipopolysaccharide (LPS) further promoted the expression levels of the inflammatory cytokines in HD11 cells. Interference with the expression of MyD88 significantly reduced the expression level of inflammatory factors in HD11 cells (P < 0.05) and had an antagonistic effect with LPS to alleviate the inflammatory reaction. In conclusion, the MyD88 gene has a pro-inflammatory effect and is highly expressed in the beak trimming and heat stress models in chicks, regulating the inflammatory response in poultry. It was involved in regulating the expression of immune-related genes in HD11 cells and had a synergistic effect with LPS., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vestigial-like ( Vgll ) genes are widespread in vertebrates and play an important role in muscle development. In this study, we used bioinformatics methods to systematically identify the chicken VGLL family in the whole genome and investigated its evolutionary history and gene structure features. Tissue expression spectra combined with real-time PCR data were used to analyze the organizational expression pattern of the genes. Based on the maximum likelihood method, a phylogenetic tree of the VGLL family was constructed, and 94 VGLL genes were identified in 24 breeds, among which four VGLL family genes were identified in the chicken genome. Ten motifs were detected in the VGLL genes, and the analysis of introns combined with gene structure revealed that the family was conserved during evolution. Tissue expression analysis suggested that the expression profiles of the VGLL family genes in 16 tissues differed between LU Shi and AA broilers. In addition, a single gene ( VGLL2 ) showed increased expression in chickens at embryonic days 10-16 and was involved in the growth and development of skeletal muscle in chickens in the embryonic stage. In summary, VGLL genes are involved in chicken muscle growth and development, which provides useful information for subsequent functional studies of VGLL genes.

Background: The characteristics of muscle fibers determine the growth and meat quality of poultry. In this study, we performed a weighted gene co-expression network analysis (WGCNA) on the muscle fiber characteristics and transcriptome profile of the breast muscle tissue of Gushi chicken at 6, 14, 22, and 30 weeks., Results: A total of 27 coexpressed biological functional modules were identified, of which the midnight blue module had the strongest correlation with muscle fiber and diameter. In addition, 7 hub genes were found from the midnight blue module, including LC8 dynein light chain 2 (DYNLL2). Combined with miRNA transcriptome data, miR-148a-3p was found to be a potential target miRNA of DYNLL2. Experiments on chicken primary myoblasts (CPMs) demonstrated that miR-148a-3p promotes the expression of myosin heavy chain (MYHC) protein by targeting DYNLL2, proving that it can promote differentiation of myoblasts., Conclusions: This study proved that the hub gene DYNLL2 and its target miR-148-3p are important regulators in chicken myogenesis. These results provide novel insights for understanding the molecular regulation mechanisms related to the development of chicken breast muscle., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

In modern poultry production, stress-induced immunosuppression leads to serious economic losses and harm to animals, but the molecular mechanisms governing the effects of stress on the chicken thymus have not been elucidated. In this study, we successfully constructed a stress model of 7-day-old Gushi chickens by adding exogenous corticosterone (CORT) to their diet and determined the microRNA (miRNA) expression profile of thymus tissues using RNA-seq technology. The results identified 51 differentially expressed miRNAs (DEMs), including 30 upregulated miRNAs and 21 downregulated miRNAs. A total of 164 target genes of the DEMs were predicted based on bioinformatic analysis methods, and Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these target genes were performed. The results from the GO enrichment analysis of the target genes identified 349 significantly enriched terms, including terms associated with the stress response and immune function that are primarily involved in the negative regulation of phagocytosis, the response to stress and the cellular response to stimulus. The KEGG pathway analysis indicated that the enriched pathways related to immunity or stress included the MAPK signaling pathway, lysosomes, endocytosis, and the RIG-I-like receptor signaling pathway. Among these pathways, DEMs (such as gga-miR-2954, gga-miR-106-5p, and gga-miR-16-5p) and corresponding target genes (such as IL11Ra, SIKE1, and CX3CL1) might be strongly correlated with thymic immunity in chickens. The results of this study provide a reference for further research on the molecular regulatory mechanisms governing the effect of stress on the immune function of the chicken thymus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Domestication and breeding have reshaped the genomic architecture of chicken, but the retention and loss of genomic elements during these evolutionary processes remain unclear. We present the first chicken pan-genome constructed using 664 individuals, which identified an additional approximately 66.5-Mb sequences that are absent from the reference genome (GRCg6a). The constructed pan-genome encoded 20,491 predicated protein-coding genes, of which higher expression levels are observed in conserved genes relative to dispensable genes. Presence/absence variation (PAV) analyses demonstrated that gene PAV in chicken was shaped by selection, genetic drift, and hybridization. PAV-based genome-wide association studies identified numerous candidate mutations related to growth, carcass composition, meat quality, or physiological traits. Among them, a deletion in the promoter region of IGF2BP1 affecting chicken body size is reported, which is supported by functional studies and extra samples. This is the first time to report the causal variant of chicken body size quantitative trait locus located at chromosome 27 which was repeatedly reported. Therefore, the chicken pan-genome is a useful resource for biological discovery and breeding. It improves our understanding of chicken genome diversity and provides materials to unveil the evolution history of chicken domestication., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Valgus-varus Deformity (VVD) leg disease often affects chickens raised in modern large-scale breeding operations. Losses due to VVD are costly to farmers, and the condition also causes significant suffering in affected birds. In this study, we profiled RNAs from the spleens of VVD (BS) and healthy (JS) broilers using high-throughput sequencing to identify miRNAs that might be involved in the development of the disease. Fifty differentially expressed miRNAs (DEMs) were found, of which 30 were up-regulated and 20 were down-regulated in VVD-affected birds (|log 2 Fold Change| ≥ 1 and q -value < 0.05). DEMs were matched with putative target genes and 864 target genes were found. Gene Ontology (GO) analyses of these target genes showed that they were significantly enriched in the "cytoplasm" term ( q -value < 0.05), and most of the target genes were enriched in "cellular component". Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that they were significantly enriched in 11 signaling pathways ( P -value < 0.05), including metabolic pathways, 2-oxocarboxylic acid metabolism, regulation of actin cytoskeleton, purine metabolism, endocytosis and so on. And we found that they were enriched in immune-related pathways in which MAPK, Notch, JAK-Stat, Toll-like receptor, p53 and other single pathways were involved in the development of skeletal diseases. Differentially expressed mRNAs obtained from our previous study were used to construct an interaction network consisting of 16 DEMs and 21 differentially expressed mRNAs (|log 2 Fold Change| ≥ 1 and q -value < 0.05). We found that miR-12247-5p, miR-15c-5p, miR-15b-5p, FKBP5 and HSP90AB1 were at the center of network interaction. This study provides a foundation for further investigations of the pathogenesis and genetic mechanisms underlying VVD.

Stress-induced immunosuppression is one of the most widespread problems in the poultry industry. Understanding the molecular regulatory mechanism of immunosuppression induced by stress in the chicken spleen would provide a scientific foundation for the prevention of stress reactions and antistress molecular breeding in poultry. To assess the protein expression profile of spleen tissue in a stress-included immunosuppression model, we performed a TMT-based proteomic analysis of chicken spleen tissue in a Dex-induced immunosuppression model (group C) and a control group (group A). We identified 590 differentially abundant proteins (DAPs) in chicken spleen tissue. These DAPs were significantly enriched in the following functional categories: ECM-receptor interaction, DNA replication, p53 signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway. Integrative analysis of the proteome and our previous transcriptome data revealed 62 DAPs showing correlations with the expression of their encoding mRNAs. Complementary proteome- and transcriptome-level analyses revealed a complex molecular network of stress-included immunosuppression. DPP4 and ALDH1A3 were the most significantly upregulated DAPs. GBP and OASL were identified as important nodes in the network related to stress-induced immunosuppression. The candidate genes identified in this study may be useful for the marker-based breeding of new chicken varieties with reduced stress levels. SIGNIFICANCE: This study provides a large amount of new information about the spleen proteome of the Dex-induced immunosuppression in chicks, as well as the correlation of transcriptome and proteome. Analysis of this resource has enabled us to examine mechanism of protein and transcript diversification, which expands the understanding of the complexity of the mechanism of stress-induced immunosuppression., (Copyright © 2021. Published by Elsevier B.V.)

With the rapid development of the poultry breeding industry and highly intensive production management, the losses caused by stress responses are becoming increasingly serious. To screen candidate genes related to chicken stress and provide a basis for future research on the molecular mechanisms governing the effects of stress on chicken immune function, we successfully constructed a chicken stress model by exogenously introducing corticosterone (CORT). RNA-seq technology was used to identify and analyze the mRNA and enrichment pathways of the thymus in the stress model group and the control group. The results showed that there were 101 significantly differentially expressed genes (SDEGs) (Padj < 0.05, |log2fold changes| ≥ 1 and FPKM >1), of which 44 were upregulated genes, while 57 were downregulated genes. Gene Ontology (GO) enrichment analysis found that the terms related to immunity or stress mainly included antigen processing and presentation, positive regulation of T cell-mediated immunity, and immune effector process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the main pathways related to immunity or stress were the PPAR signaling pathway, NOD-like receptor signaling pathway, and intestinal immune network for IgA production. Among the SDEGs, XCL1, HSPA8, DMB1 and BAG3 are strongly related to immunity or stress and may be important genes involved in regulating stress affecting the immune function of chickens. The above results provide a theoretical reference for subsequent research on the molecular regulatory mechanisms by which stress affects the immune function of poultry., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

For investigating the effects of stress on the immune response of chickens, we established a corticosterone (CORT)-induced stress model by exogenous intake of CORT. Control group was fed with a basal diet and the stress model group was fed with a 30 mg/Kg CORT-treated diet in ad libitum conditions for 7 days. Then, we used RNA-seq technology to identify the expression pattern of miRNAs, target genes, and relevant pathways in chicken spleen. Results showed that 71 differentially expressed miRNAs (DEMs) were determined, 9 of which were significantly differentially expressed miRNAs (SDEMs), and 241 target genes of DEMs were predicted. GO annotation and KEGG pathway analysis were carried out to understand the role of the DEMs. Out of 287 significantly enriched GO terms, 37 were stress- or immune-related, such as response to light stimulus, detection of oxidative stress, and immune response in mucosal-associated lymphoid tissue. Out of 85 KEGG pathways, 8 were related to stress or immunity, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, and RLR signaling pathway. We then constructed the interaction networks between target genes from immune-related pathways and their DEMs. The analysis results suggested that some DEMs (gga-miR-17 family, gga-miR-15/16 family, gga-miR-2954 and gga-miR-34b-5p) and target genes (SIKE1, CX3CL1, IL11Ra, PIGR, and CDKN1A) were core miRNAs and genes. This study revealed the dynamic miRNA transcriptome, target genes and related pathways in chicken spleen under CORT-induced stress model, which provided a basis for studying the molecular mechanism of stress affecting immune function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chicken growth traits are economically important, but the relevant genetic mechanisms have not yet been elucidated. Herein, we performed a genome-wide association study to identify the variants associated with growth traits. In total, 860 chickens from a Gushi-Anka F 2 resource population were phenotyped for 68 growth and carcass traits, and 768 samples were genotyped based on the genotyping-by-sequencing (GBS) method. Finally, 734 chickens and 321,314 SNPs remained after quality control and removal of the sex chromosomes, and these data were used to carry out a GWAS analysis. A total of 470 significant single-nucleotide polymorphisms (SNPs) for 43 of the 68 traits were detected and mapped on chromosomes (Chr) 1-6, -9, -10, -16, -18, -23, and -27. Of these, the significant SNPs in Chr1, -4, and -27 were found to be associated with more than 10 traits. Multiple traits shared significant SNPs, indicating that the same mutation in the region might have a large effect on multiple growth or carcass traits. Haplotype analysis revealed that SNPs within the candidate region of Chr1 presented a mosaic pattern. The significant SNPs and pathway enrichment analysis revealed that the MLNR, MED4, CAB39L, LDB2, and IGF2BP1 genes could be putative candidate genes for growth and carcass traits. The findings of this study improve our understanding of the genetic mechanisms regulating chicken growth and carcass traits and provide a theoretical basis for chicken breeding programs.

Many studies have shown that the LDB2 gene plays a regulatory role in retinal development and the cell cycle, but its biological role remains unclear. In this study, a 31-bp indel in the LDB2 gene was found for the first time on the basis of 2797 individuals from 10 different breeds, which led to different genotypes among individuals (II, ID and DD). Among these genotypes, DD was the most dominant. Association analysis of an F 2 resource population crossed with the Gushi (GS) chicken and Anka chicken showed that the DD genotype conferred a significantly greater semi-evisceration weight (SEW, 1108.665 g ± 6.263), evisceration weight (EW, 927.455 g ± 5.424), carcass weight (CW, 1197.306 g ± 6.443), breast muscle weight (BMW, 71.05 g ± 0.574), and leg muscle weight (LMW, 100.303 g ± 0.677) than the ID genotype (SEW, 1059.079 g ± 16.86; EW, 879.459 g ± 14.446; CW, 1141.821 g ± 17.176; BMW, 67.164 g ± 1.523; and LMW, 96.163 g ± 1.823). In addition, LDB2 gene expression in different breeds was significantly higher in the breast muscles and leg muscles than in other tissues. The expression level in the breast muscle differed significantly among stages of GS chicken development, with the highest expression observed at 6 weeks. The expression levels in the pectoral muscles differed significantly among Ross 308 genotypes. In summary, we studied the relationships between a 31-bp indel in the LDB2 gene and economic traits in chickens. The indel was significantly correlated with multiple growth and carcass traits in the F 2 resource population and affected the expression of the LDB2 gene in muscle tissue. In short, our study revealed that the LDB2 gene 31-bp indel can be used as a potential genetic marker for molecular breeding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Valgus-varus Deformity (VVD) is an outward or inward deviation of the tibiotarsus or tarsometatarsus, which results in physical distress of chickens and economic loss in poultry industry. While the etiology and pathogenesis of VVD at the molecular level are still not fully understood so far. Here, based on a case/control design with VVD birds and normal birds, we identified genes and lncRNAs which associated with VVD using RNA sequencing. Transcriptome analysis revealed 231 differentially expressed mRNAs and 23 differentially expressed lncRNAs between case and control of leg cartilage. We identified the cis- and trans-regulatory targets of the differentially expressed lncRNAs, and we constructed a functional lncRNA-mRNA co-expression network. Analysis of the network showed that the differentially expressed mRNAs and the target genes of the differentially expressed lncRNAs were enriched in the signaling pathways associated with bone development, including p53, MAPK, Toll-like receptor, Jak-STAT, Hedgehog, and PPAR. The expression levels of DENND4A, FGF10, FGF12 and BMP3 were also determined in cartilage and other six tissues. Overall, our study predicted the mRNAs and lncRNAs related with leg diseases by transcriptome analyses, which might contribute to understand the etiology and pathogenesis of VVD. It established the foundation for the further research on the function of -mRNAs and lncRNAs in skeleton development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

MiRNAs are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. SNPs in miRNA genes may lead to phenotypic variation by altering miRNA expression and their targets. In this study, miR-1704 expression profiles in nine tissues at 1 d, 6 weeks and 16 weeks old Gushi chickens were detected. MiR-1704 was widely expressed in the detection of tissues. The expression in 1 d and 6 weeks old was low abundance, while its expression at 16 weeks was very high. An rs14668705 (C > G) SNP was detected within the pre-miR-1704 in an F 2 resource population of Gushi chicken crossed with Anka broiler. Bioinformatic analysis indicated that the C > G mutation could introduce a base-pair mismatch and cause the change of free energy. Experiments further revealed that the rs14668705 in precursor miR-1704 could significantly affect mature miR-1704 biogenesis and was significantly associated with body weight at the age of 0, 6, 8, 10, and 12 weeks, shank circumference at 4, 8, and 12 weeks, carcass weight, and semi-evisceration weight ( p  < 0.05). Insulin receptor 2 ( IRS2 ) gene, one of the potential targets of miR-1704 was identified and further confirmed. These data suggested that miR-1704 targeted IRS2 and have an effect on body weight in chicken.

Stress-induced immunosuppression is a common problem in the poultry industry, but the specific mechanism of its effect on the immune function of chicken has not been clarified. In this study, 7-day-old Gushi cocks were selected as subjects, and a stress-induced immunosuppression model was successfully established via daily injection of 2.0 mg/kg (body weight) dexamethasone. We characterized the spleen transcriptome in the control (B&#95;S) and model (D&#95;S) groups, and 515 significant differentially expressed genes (SDEGs) (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) > 1, adjusted p -value (padj) < 0.05 and Fold change (|FC|) ≥ 2) were identified. The cytokine-cytokine receptor interaction signaling pathway was identified as being highly activated during stress-induced immunosuppression, including the following SDEGs- CXCL13L2, CSF3R, CSF2RB, CCR9, CCR10, IL1R1, IL8L1, IL8L2, GHR, KIT, OSMR, TNFRSF13B, TNFSF13B , and TGFBR2L . At the same time, immune-related SDEGs including CCR9 , CCR10, DMB1, TNFRSF13B, TNFRSF13C and TNFSF13B were significantly enriched in the intestinal immune network for the IgA production signaling pathway. The SDEG protein-protein interaction module analysis showed that CXCR5, CCR8L, CCR9, CCR10, IL8L2, IL8L1, TNFSF13B, TNFRSF13B and TNFRSF13C may play an important role in stress-induced immunosuppression. These findings provide a background for further research on stress-induced immunosuppression. Thus, we can better understand the molecular genetic mechanism of chicken stress-induced immunosuppression.

An increasing number of studies have shown that quantitative trait loci (QTLs) at the end of chromosome 1 identified in different chicken breeds and populations exert significant effects on growth traits in chickens. Nevertheless, the causal genes underlying the QTL effect remain poorly understood. Using an updated gene database, a novel lncRNA (named LncFAM) was found at the end of chromosome 1 and located in a growth and digestion QTL. This study showed that the expression level of LncFAM in pancreas tissues with a high weight was significantly higher than that in pancreas tissues with a low weight, which indicates that the expression level of LncFAM was positively correlated with various growth phenotype indexes, such as growth speed and body weight. A polymorphism screening identified four polymorphisms with strong linkage disequilibrium in LncFAM: a 5-bp indel in the second exon, an A/G base mutation, and 7-bp and 97-bp indels in the second intron. A study of a 97-bp insertion in the second intron using an F2 chicken resource population produced by Anka and Gushi chickens showed that the mutant individuals with genotype II had the highest values for body weight (BW) at 0 days and 2, 4, 6, 8, 10 and 12 weeks, shank girth (SG) at 4, 8 and 12 weeks, chest width (CW) at 4, 8 and 12 weeks, body slant length (BSL) at 8 and 12 weeks, and pelvic width (PW) at 4, 8 and 12 weeks, followed by ID and DD genotypes. The amplification and typing of 2,716 chickens from ten different breeds, namely, the F2 chicken resource population, dual-type chickens, including Xichuan black-bone chickens, Lushi green-shell layers, Dongxiang green-shell layers, Changshun green-shell layers, and Gushi chickens, and commercial broilers, including Ross 308, AA, Cobb and Hubbard broilers, revealed that II was the dominant genotype. Interestingly, only genotype II existed among the tested populations of commercial broilers. Moreover, the expression level in the pancreas tissue of Ross 308 chickens was significantly higher than that in the pancreas tissue of Gushi chickens (P < 0.001), which might be related to the conversion rates among different chickens. The prediction and verification of the target gene of LncFAM showed that LncFAM might regulate the expression of its target gene FAM48A through cis-expression. Our results provide useful information on the mutation of LncFAM, which can be used as a potential molecular breeding marker., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Carboxyl ester lipase (CEL) encodes a cholesterol ester hydrolase that is secreted into the duodenum as a component of pancreatic juice. The objective of this study was to characterize the CEL gene, investigate the association between the CEL promoter variants and chicken phenotypic traits, and explore the CEL gene regulatory mechanism. An insertion/deletion (indel) caused by a 99-bp insertion fragment was shown for the first time in the chicken CEL promoter, and large differences in allelic frequency were found among commercial breeds, indigenous and feral birds. Association analysis demonstrated that this indel site had significant effects on shank length, shank girth, chest breadth at 8 weeks (p < 0.01), evisceration weight, sebum weight, breast muscle weight, and leg weight (p < 0.05). Tissue expression profiles showed extremely high levels of the CEL gene in pancreatic tissue. Moreover, the expression levels of the genes APOB, MTTP, APOV1 and SREBF1, which are involved in lipid transport, were significantly reduced by adding a 4% oxidized soybean oil diet treatment at the individual level and transfecting the embryonic primary hepatocytes with a CEL-overexpression vector. Interestingly, the results showed that the expression level of the II homozygous genotype was significantly higher than that of the ID and DD genotypes, while individuals with DD genotypes had higher phenotypic values. Therefore, these data suggested that the CEL gene might affect body growth by participating in hepatic lipoprotein metabolism and that the 99-bp indel polymorphism could be a potentially useful genetic marker for improving the economically important traits of chickens.

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Abdominal fat is the major adipose tissue in chickens. The growth status of abdominal fat during postnatal late development ultimately affects meat yield and quality in chickens. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression at the post-transcriptional level. Studies have shown that miRNAs play an important role in the biological processes involved in adipose tissue development. However, few studies have investigated miRNA expression profiles and their interaction networks associated with the postnatal late development of abdominal adipose tissue in chickens., Results: We constructed four small RNA libraries from abdominal adipose tissue obtained from Chinese domestic Gushi chickens at 6, 14, 22, and 30 weeks. A total of 507 known miRNAs and 53 novel miRNAs were identified based on the four small RNA libraries. Fifty-one significant differentially expressed (SDE) miRNAs were identified from six combinations by comparative analysis, and the expression patterns of these SDE miRNAs were divided into six subclusters by cluster analysis. Gene ontology enrichment analysis showed that the SDE miRNAs were primarily involved in the regulation of fat cell differentiation, regulation of lipid metabolism, regulation of fatty acid metabolism, and unsaturated fatty acid metabolism in the lipid metabolism- or deposition-related biological process categories. In addition, we constructed differentially expressed miRNA-mRNA interaction networks related to abdominal adipose development. The results showed that miRNA families, such as mir-30, mir-34, mir-199, mir-8, and mir-146, may have key roles in lipid metabolism, adipocyte proliferation and differentiation, and cell junctions during abdominal adipose tissue development in chickens., Conclusions: This study determined the dynamic miRNA transcriptome and characterized the miRNA-mRNA interaction networks in Gushi chicken abdominal adipose tissue for the first time. The results expanded the number of known miRNAs in abdominal adipose tissue and provide novel insights and a valuable resource to elucidate post-transcriptional regulation mechanisms during postnatal late development of abdominal adipose tissue in chicken.

The large economic losses caused by leg disorders have raised concerns in the broiler industry. Several types of leg disorders in broilers have been identified, such as tibial dyschondroplasia (TD), femoral head necrosis (FHN), and valgus-varus deformity (VVD). In this study, phenotypic changes associated with VVD were examined using clinical diagnosis, anatomical examination, measured growth performance, bone traits, and serum indicators. The incidence of VVD among the chicken population at a commercial facility in Tangshan China was 1.75% (n = 52,000), distributed about 1:1 (n = 122), between females and males. A majority of chickens were characterized by a unilaterally abnormality, while appropriately 17.6% by bilateral abnormality. Approximately 97.9% of affected broilers were classified as the "valgus" type. Growth traits, including body weight, shank length, and shank girth, were significantly lower in chickens with VVD, while tibia and metatarsal bone indexes were about 1.3-fold higher in the affected birds than in the normal birds. Bone mineral density, bone breaking strength, and several serum indicators were significantly different between affected and normal broilers. Sparse and disarranged bony trabecular was observed in abnormal broilers by histological analysis. Generally, leg disorders are associated with compromised growth, bone quality, bone structure, and lipid metabolism. This study provides a reference for clinical diagnosis of VVD and lays a foundation for exploring its underlying mechanisms., (© 2019 Poultry Science Association Inc.)

Intramuscular fat (IMF) traits are important factors that influence meat quality. However, the molecular regulatory mechanisms that underlie this trait in chickens are still poorly understood at the gene coexpression level. Here, we performed a weighted gene coexpression network analysis between IMF traits and transcriptome profile in breast muscle in the Chinese domestic Gushi chicken breed at 6, 14, 22, and 30 weeks. A total of 26 coexpressed gene modules were identified. Six modules, which included the dark gray, purple, cyan, pink, light cyan, and blue modules, showed a significant positive correlation (P < 0.05) with IMF traits. The strongest correlation was observed between the dark gray module and IMF content (r = 0.85; P = 4e-04) and between the blue module and different fatty acid content (r = 0.87~0.91; P = 5e-05~2e-04). Enrichment analysis showed that the enrichment of biological processes, such as fatty acid metabolic process, fat cell differentiation, acylglycerol metabolic process, and glycerolipid metabolism were significantly different in the six modules. In addition, the 32, 24, 4, 7, 6, and 25 hub genes were identified from the blue, pink, light cyan, cyan, dark gray, and purple modules, respectively. These hub genes are involved in multiple links to fatty acid metabolism, phospholipid metabolism, cholesterol metabolism, diverse cellular behaviors, and cell events. These results provide novel insights into the molecular regulatory mechanisms for IMF-related traits in chicken and may also help to uncover the formation mechanism for excellent meat quality traits in local breeds of Chinese chicken., (© 2019 Wiley Periodicals, Inc.)

Previous studies have revealed a significant association between SNPs found within the heparan sulfate 6-O-sulfotransferase 3 ( HS6ST3 ) gene and obesity. This study identified a novel 43-bp indel polymorphism in intron 1 of HS6ST3 in 1963 chickens from nine different breeds, and three genotypes, designated II, ID and DD, were observed. The frequency of the 'I' (0.62-0.87) allele was higher than that of the 'D' (0.13-0.38) allele. A total of 777 individuals of the Gushi-Anka F 2 resource population were used for the analysis of associations according to growth traits, carcass traits, serum variables and meat quality traits. The results showed that the 43-bp indel polymorphism was significantly associated with the body weight at 4 and 6 weeks of age, chest depth at 4 and 12 weeks of age and shank girth at 12 weeks of age ( P  < 0.05). In terms of the carcass traits, the indel polymorphism was significantly associated with breast muscle weight, heart weight and leg weight (P < 0.05). These findings suggested that this indel polymorphism has the potential to become a new target for the marker-assisted selection of chicken growth and carcass traits.

Zinc finger proteins are a class of transcription factors with finger-like domains and have diverse uses in biological processes, including development, differentiation, and metabolism. In this study, we identified the absence of the 24 bp sequence in the third exon of the zinc finger protein 764-like (ZNF764L) gene that lead to the production of two new transcripts, ZNF764L-SV1 and ZNF764L-SV2, and the sum of the expression levels of the two transcripts is approximately equal the total RNA expression level. Temporal and spatial expression showed that ZNF764L had higher expression during the embryonic stage. Moreover, the research study revealed a 22-bp indel mutation in the first exon region of ZNF764L gene. Statistically significant results (P < 0.05) were encountered for this indel for chicken growth and carcass traits, which include birth weight, chest breadth and body slanting length at 4 weeks of age and subcutaneous fat weight and others. Genetic parameter analysis showed that D is the predominant allele in the commercial chicken population. Gene expression for each genotype showed that birds carrying the II allele had a higher expression level than the other genotypes. These findings enrich the understanding of ZNF764L gene function and enhance reproduction in the chicken industry., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Many studies have reported that cyclin-dependent kinase inhibitor 3 (CDKN3) is involved in the cell cycle. However, the function of CDKN3 has not been well elucidated in organisms. In this study, a multiallelic indel caused by a 19-bp fragment and a 2 × 19 bp fragment was shown for the first time to be inserted into the promoter of the CDKN3 gene in 1994 chickens from 9 different breeds. In addition, 6 genotypes (C5C5, C4C4, C3C3, C4C5, C3C4, and C3C5) were observed (C3C3, C4C4, C5C5 have 3 × 19 bp, 4 × 19 bp, and 5 × 19 bp, respectively). Among these genotypes, the C4C4 genotype was the most dominant genotype in 9 breeds. The results of χ2 analysis of CDKN3 gene in different breeds showed that there were significant differences in the distribution of genotypes among different cultivars (P < 0.01). In addition, association study with F2 chicken resource population which produced by Anka and Gushi chickens showed that the C3C4 genotypes had the greatest semi-evisceration weight (SEW, 1163.94 ± 46.84), evisceration weight (EW, 964.15 ± 41.16), head weight (HW, 45.55 ± 1.43), claw weight (CW, 63.42±2.86), wing weight (WW, 129.15±5.48), liver weight (LW, 29.96±1.27), carcass weight (cW, 1286.96±49.53), weight at 10 (1190.68±45.68) and 12 (1430.65±54.45) wk, followed by C3C3, C4C4, C5C5, C4C5, whereas C3C5 genotypes having the lowest SEW (989.21±47.71), EW (841.38±40.55), HW (41.03±1.46), CW (54.36±2.81), WW (116.31±5.39), LW (27.31±1.25), cW (1093.29±49.99), weight at 10 (1036.10±44.99) and 12 (1246.28±53.59) wk. Expression levels of CDKN3 in breast muscle of chickens with C4C4 (0.72±0.02), C3C3 (0.95±0.41), and C4C5 (0.74±0.13) genotypes were significantly lower than those with C5C5 (1.80±0.01) and C3C5 (2.14±0.17) genotypes (P < 0.05). In conclusion, we investigated the effect of a multiallelic indel in the CDKN3 gene on the economic traits of chickens, and this indel was significantly associated with growth and carcass traits in chickens. Collectively, our findings provide useful information about the repeat sequence indel in the promoter region of the CDKN3 gene as a potential molecular marker for chicken breeding., (© 2018 Poultry Science Association Inc.)

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&#95;B group) and control group (basal diet; B&#95;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&#95;B group compared with the B&#95;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.

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 biological functions of long noncoding RNAs (lncRNAs), which play an important role in regulating development and gene expression, may be affected by variations in lncRNA gene loci or associated genomic sequences. However, the functions of many lncRNAs remain unknown. To analyse correlations between mutations in pouMU1 with chicken growth and carcass traits, 860 chickens from a Gushi×Anka F2 resource population and 96 Lushi, Xichuan, Changshun and recessive white chickens were used to evaluate the genetic effect of the pouMU1 gene. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to analyse the relative expression levels of pouMU1 in nine different tissues and stages of development. pouMU1 expression was highest in pectoralis and leg muscles, whereas no expression was observed in the heart, liver and abdominal fat. Using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, two novel sequence mutations (g.1198A>G and g.1238-1239del/insGA) were detected in the pouMU1 gene. SPSS software was used for statistical analysis in association studies. Based on the association data, the presence of both variants was significantly associated with leg muscle fibre width and leg muscle fibre roundness (P < 0.05) and highly associated with leg muscle fibre girth and body weight at 0 week of age (P < 0.01). These data suggest that pouMU1 might participate in regulating chicken muscle development and growth, and the findings offer new insight into the functions of sequence mutations in lncRNAs.

Background: 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., Results: 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., Conclusion: 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.

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R-MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17β-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Laying performance is an important economic trait in hens, and this physiological process is largely influenced by the liver function. The livers of hens at 20- and 30-week-old stages were investigated using the next generation sequencing to identify the differences of microRNA expression profiles. Compared with the 20-week-old hens, 67 down- and 13 up-regulated microRNAs were verified to be significant differentially expressed (false discovery rate, FDR ≤ 0.05) (SDE) in the 30-week-old. We also identified 13 down- and 6 up-regulated novel differentially expressed (DE) microRNAs. miR-22-3p and miR-146b-5p, which exhibit critical roles in mammalian lipid metabolism, showed the most abundant expression and the highest fold-change, respectively. A total of 648 potential target genes of the SDE microRNAs were identified through an integrated analysis of microRNAs and the DE genes obtained in previous RNA-sequencing, including FADS1, FADS2, ELOVL6 and ACSL5, which are critical lipid metabolism-related regulators. Bioinformatic analyses revealed that target genes were mainly enriched in lipid-related metabolism processes. This work provides the first study of the expression patterns of hepatic microRNAs between 20- and 30-week old hens. The findings may serve as a fundamental resource for understanding the detailed functions of microRNAs in the molecular regulatory systems of lipid metabolism.

Genetic variations in microRNAs (miRNAs) including primary miRNAs, precursor miRNAs and mature miRNAs can lead to phenotypic variation by altering the biogenesis of miRNAs and/or their binding to target mRNAs. Increasing functional studies suggest that polymorphisms occurring in miRNAs can lead to phenotypic variation in farm animal. Here, we identified a single nucleotide polymorphism (SNP) located in the precursor of chicken miRNA-1606 gene. The association study on body indexes, body weight at different growth stages, and carcass traits was performed in a Gushi-Anka F2 population resource. The SNP was not only significantly associated with body weight at 10 and 12 weeks, respectively, but also with chicken shank length, chest depth and body slanting length at 8 weeks; shank length, pectoral angle, body slanting length and pelvis breadth at 12 weeks, respectively. And the polymorphism was also significantly associated with carcass traits including semi-evisceration weight, evisceration weight, breast muscle weight, leg weight and carcass weight as well. The observed values of individuals with CA genotype were significantly higher than CC genotype both in body weight at different stages and carcass traits. This SNP altered the predicted second structure of pre-mir-1606, with the altering of the free energy values. And the relative expression level of mature miRNA between CA and AA was significantly changed in leg muscle. Our data suggested that miRNA-1606 may be a candidate gene associated with chicken growth traits., (Copyright © 2015 Elsevier B.V. All rights reserved.)