Integrated analysis of microRNA and mRNA interactions in ovary of counter-season breeding and egg-ceased geese (Anser cygnoides).

Egg-ceasing is a phenomenon that occurs in most avian species and significantly reduces productivity. Although several factors are reported to regulate the reproduction progress, the underlying molecular mechanism of egg-ceasing remains obscure. Herein, we identified and explored the differentially expressed miRNAs and mRNAs involved in ovarian atrophy via high throughput sequencing. We identified a total of 901 mRNAs and 50 miRNAs that were differentially expressed in egg-laying and atrophic ovaries. Among them, numerous differentially expressed gene (DEG) transcripts and target genes for miRNAs were significantly enriched in Gene Ontology terms such as reproductive processes, cell proliferation, and apoptosis pathways. In addition, an interaction network was constructed by considering target relationships and correlation of the expression levels between ovary development-related genes, miRNAs and pathways. We discovered mRNA and miRNAs transcripts that are candidate regulators of ovary development in egg-ceased geese. Our findings expanded our understanding of the functional of miRNAs in ovarian atrophy and demonstrated that RNA-Seq is a powerful tool for examining the molecular mechanism in regulating egg-ceasing.

Genotype frequency distributions of 28 SNP markers in two commercial lines and five Chinese native chicken populations.

BACKGROUND: Modern breeding in the poultry industry mainly aims to produce high-performance poultry lines and breeds in two main directions of productivity, meat and eggs. To understand more about the productive potential of lowly selected Chinese native chicken populations, we selected 14 representative SNP markers strongly associated with growth traits or carcass traits and 14 SNP markers strongly associated with egg laying traits through previous reports. By using the MassArray technology, we detected the genotype frequency distributions of these 28 SNP markers in seven populations including four lowly selected as well as one moderately selected Sichuan native chicken populations, one commercial broiler line and one commercial layer line. RESULTS: Based on the genotype frequency distributions of these 28 SNP markers in 5 native chicken populations and 2 commercial lines, the results suggested that these Chinese indigenous chicken populations have a relatively close relationship with the commercial broiler line but a marked distinction from the commercial layer line. Two native chicken breeds, Shimian Caoke Chicken and Daheng Broilers, share similar genetic structure with the broiler line. CONCLUSIONS: Our observations may help us to better select and breed superior domestic chickens and provide new clues for further study of breeding programs in local chicken populations.

Polymorphism in MC1R, TYR and ASIP genes in different colored feather chickens.

Coat color genetics successfully adapted and applied to different animal species, which provides a good demonstration of the concept of comparative genetics. In this study, we sequenced 945 bp fragments of melanocortin 1 receptor (MC1R) gene, 421 bp fragments of exon 1 of tyrosinase (TYR) gene and 266 bp fragments of exon 3 of agouti signaling protein (ASIP) gene for 250 individuals with five plumage color patterns. We detected a total of three SNPs (T398A, T637C, and G920C) in MC1R and built six haplotypes (H1-H6) based on the three SNPs. H5 and H6 haplotypes were mainly concentrated in white and grey chicken. And diplotypes H2H3 occurred in white feather and black-speckle feather with the same frequency. Moreover, a total of three SNPs (C47G, T120C, and T172C) in TYR were found and built six haplotypes (P1-P6) based on the three SNPs. Among them, haplotype P2, P3 and P6 were not occurred in black chicken, the diplotypes P1P6 and P4P6 were only distributed in white, gray and black-speckled feather. We only detected one SNP (T168C) in ASIP gene and found that genotype TT was advantage genotype in the different plumage color groups of chickens. Collectively, our study suggested an association between plumage color and genetic variation of MC1R, TYR and ASIP in chicken.

[Genetic regulation of broodiness in poultry].

Broodiness is a behavior commonly occurring in the poultry industry, which is characterized by inappetence, egg-laying cessation and incubation. Different from laying fowls, the ovary and oviduct of broodiness fowls is degenerate. Broodiness is a low heritability trait, which is controlled by multiple genes in autosomes and influenced by three factors, including environment, endocrine and genetics. In addition to the observation of behavioral characteristics, the current research of broodiness focuses on evaluating the genetic mode, endocrine factors, nesting candidate genes and their polymorphisms in poultry. Given the rapid development of high-throughput sequencing, the joint analyses of genomics, transcriptomics and proteomics have been used to screen out the candidate genes, pathways and molecular mechanism of broodiness. In this review, we summarize the candidate genes, microRNAs and pathways involved in broodiness to provide a reference for further research on poultry broodiness.

KLF5 functions in proliferation, differentiation, and apoptosis of chicken satellite cells.

KLF5 is an important regulator of cell proliferation, differentiation, and apoptosis in mammals. Little is known about the function of KLF5 in the regulation of chicken. Hence, qPCR was used to detect the expression of KLF5 in different tissues of chicken. And chicken skeletal muscle satellite cells (SMSCs) were transfected KLF5-specific small interfering RNA (siRNA) to assay SMSCs' proliferation, differentiation, and apoptosis. The results showed that KLF5 expressed higher in skeletal muscle than in the other tissues of chicken. Knockdown of KLF5 significantly inhibited the differentiation and increased apoptosis of chicken SMSCs, but it had no significant effect on proliferation of SMSCs. These results indicate that KLF5 plays an essential role during myogenesis, which will affect muscle repair and muscle regeneration, and may ameliorate muscle aging or sarcopenia.

Molecular Characterization, Expression and Functional Analysis of Chicken STING.

Innate immunity is an essential line of defense against pathogen invasion which is gained at birth, and the mechanism involved is mainly to identify pathogen-associated molecular patterns through pattern recognition receptors. STING (stimulator of interferon genes) is a signal junction molecule that hosts the perception of viral nucleic acids and produces type I interferon response, which plays a crucial role in innate immunity. However, relatively few studies have investigated the molecular characterization, tissue distribution, and potential function of STING in chickens. In this study, we cloned the full-length cDNA of chicken STING that is composed of 1341 bp. Sequence analyses revealed that STING contains a 1140-bp open-reading frame that probably encodes a 379-amino acid protein. Multiple sequence alignments showed that the similarity of the chicken STING gene to other birds is higher than that of mammals. Real-time polymerase chain reaction (PCR) assays revealed that STING is highly expressed in the spleen, thymus and bursa of fabricious in chickens. Furthermore, we observed that STING expression was significantly upregulated both in vitro and in vivo following infection with Newcastle disease virus (NDV). STING expression was also significantly upregulated in chicken embryo fibroblasts upon stimulation with poly(I:C) or poly(dA:dT). Taken together, these findings suggest that STING plays an important role in antiviral signaling pathways in chickens.

Molecular characterization of a novel ovodefensin gene in chickens.

Host defense peptides (HDPs) represent a large group of diverse small peptides that play important roles in host defense and disease resistance. In vertebrates, one of the main types of HDPs belong to defensins, which are less than 100 amino acid residues and characterized by a highly conserved motif of cysteine residues. Recently, a subfamily of defensins, namely ovodefensins (OvoDs), has been identified in birds and reptiles. However, both their family members and evolutionary relationships remain unclear. In the present study, we cloned and characterized a novel gene namely OvoDBß in chickens. Our results showed that the full length of chicken OvoDBß mRNA contains 344 bp nucleotides and encodes a 61-amino acid protein. We further revealed that the mRNA of OvoDBß is abundant in the oviduct of laying hens but absent in many other tissues. Additionally, sequences comparison and analyses suggested that OvoDBß is orthologous to the gene previously known as zebra finch OvoDB1, albeit it might exhibit specific structures. Furthermore, both OvoDBα and OvoDBß were existent in the genome of each bird, implying that two types of OvoDBs sharing same cysteine motif have already emerged before the species divergence. More importantly, recombinant OvoDBß mature peptide exerted antibacterial activity against Escherischia coli (CICC23657 strain) in vitro. These results collectively indicated that the putative sequence, namely chicken OvoDBß, is a function gene with potential antimicrobial property. Discovery and function characterization of novel HDP genes may help us develop novel antimicrobial agents in the future.

Genotypes of IFIH1 and IFIT5 in seven chicken breeds indicated artificial selection for commercial traits influenced antiviral genes.

Innate immunity is the first line against the invasion of pathogenic microorganisms. Previous reports only demonstrated production traits of commercial importance were often negatively correlated with innate disease resistance. However, whether different purpose of artificial selection influences innate immunity have not been understood. In this study, we cloned exon1, exon6 of IFIH1 and exon2 of IFIT5 by molecular biology techniques in seven different chicken breeds to detect the potential effect of artificial selection for commercial traits on disease resistance for the first time. In total, 8 single nucleotide polymorphisms(SNPs) of IFIH1 gene exon1 and exon6, 19 SNPs of IFIT5 gene exon2 were detected. We found all native chicken breeds had a relatively close relationship to broiler breeds but a remote relationship to layer breed. A great difference between CB and LLH with different selected purpose were observed. The allele frequencies of these two positive antiviral genes were associated with different purpose of artificial selection. Our experiment constituted the foundation for the interaction between commercial traits and immune trait.

Experimental Verification of CAPN1 and CAST Gene Polymorphisms in Different Generations of Da-Heng Broilers.

The micromolar calcium activated neutral protease (CAPN1) and calpastatin (CAST) have been widely regarded as genes related to muscle growth and meat tenderness. The objective of this study was to verify the association of SNPs of CAPN1 and CAST genes with carcass and tenderness traits and search the possible change patterns of SNPs in CAPN1 and CAST genes in six generations of broiler breeding process for growth rate, efficiency, and reproduction, during the third generation and the ninth generation, respectively. We found that, for CAPN1, genetic effects between SNPs (G3535A, C7198A) and meat tenderness were similar in different generations, while SNP3 (G7324A) was a novel polymorphism and had significant association with carcass and tenderness traits (P < 0.05) in this study. Furthermore, there was significant association between SNP4 (G9950A) and carcass indexes instead of tenderness traits (P < 0.05) which was consistent in the two generations. Moreover, although SNP6 (G37868A) of CAST had no relevance to carcass traits or tenderness traits in the third generation, it showed significant association with LW and CW in the ninth generation (P < 0.05).

The Relationship between MC1R Mutation and Plumage Color Variation in Pigeons.

The polymorphisms of MC1R gene play a crucial role in coat color variation in mammals; however, the relationship is still unclear in pigeons. In this study, we sequenced 741 bp fragment of the MC1R for 39 individuals with five plumage color patterns (gray plumage, n = 12; black plumage, n = 9; white plumage, n = 3; spotted plumage, n = 12; red plumage, n = 3). A total of three single nucleotide polymorphisms (SNPs) were detected, including G199A, G225A, and A466G, which subsequently determined four haplotypes (H1-H4). Among them, H1 is the predominant haplotype. Association analysis revealed that H1 and H3 were significantly associated with the black plumage trait (P < 0.05), while the H4 was significantly associated with gray plumage trait (P < 0.05). Furthermore, only diplotype H1H1 was significantly associated with black and gray traits of pigeons. Collectively, our study suggested an association between genetic variation of MC1R and plumage color in pigeon.

MyD88 Polymorphisms and Association with Susceptibility to Salmonella Pullorum.

Myeloid differentiation primary response gene 88 (MYD88), a universal adapter protein, plays an important role in activating the nuclear factor-κB (NF-κB) and regulating the expression of proinflammatory genes like tumor necrosis factor (TNF) and interleukin-1 (IL-1), which were highly involved in Salmonella Pullorum infection. To detect the relationship between polymorphisms of the MyD88 gene and Salmonella Pullorum disease, we screened the coding region (CDS) of the MYD88 gene by DNA pool construction and sequencing based on case-control study. Eight single nucleotide polymorphisms (SNPs) in the sequenced fragment (5 exons), 7 known loci and one novel mutation named G4810372T (SNP8), were found in the fifth exon. In addition, we found 7 nonsynonymous substitutions. The allele frequency of only one SNP, g.4810191C > T (SNP1), was significantly different (P < 0.05) between case and control groups. The genotype frequencies of SNP1 (g.4810191C > T) and SNP3 (g.4810257G > T) were of significant difference between the case and the control groups (P < 0.05). Collectively, SNPs of the MyD88 gene were significantly associated with susceptibility to Salmonella Pullorum infection, which can be used as a disease-resistant marker in chicken. These results provided a theoretical basis for future research on chicken breeding by marker-assisted selection.