Skeletal muscle fibers are primarily categorized into oxidative and glycolytic fibers, and the ratios of different myofiber types are important factors in determining livestock meat quality. However, the molecular mechanism for determining muscle fiber types in chickens was hardly understood. In this study, we used RNA sequencing to systematically compare mRNA and microRNA transcriptomes of the oxidative muscle sartorius (SART) and glycolytic muscle pectoralis major (PMM) of Chinese Qingyuan partridge chickens. Among the 44,705 identified mRNAs in the two types of muscles, 3,457 exhibited significantly different expression patterns, including 2,364 up-regulated and 1,093 downregulated mRNAs in the SART. A total of 698 chicken miRNAs were identified, including 189 novel miRNAs, among which 67 differentially expressed miRNAs containing 42 up-regulated and 25 downregulated miRNAs in the SART were identified. Furthermore, function enrichment showed that the differentially expressed mRNAs and miRNAs were involved in energy metabolism, muscle contraction, and calcium, peroxisome proliferator-activated receptor (PPAR), insulin and adipocytokine signaling. Using miRNA-mRNA integrated analysis, we identified several candidate miRNA-gene pairs that might affect muscle fiber performance, viz, gga-miR-499-5p/SOX6 and gga-miR-196-5p/CALM1, which were supported by target validation using the dual-luciferase reporter system. This study revealed a mass of candidate genes and miRNAs involved in muscle fiber type determination, which might help understand the molecular mechanism underlying meat quality traits in chickens. Improving meat quality has long been a goal of broiler breeding programs, especially for Chinese native breeds 1,2. However, meat quality is difficult to define because it is a complex trait influenced by numerous factors 3. As the main tissue determining meat quality, skeletal muscle is a heterogeneous tissue composed of different types of muscle fibers, varying in their biochemical and structural characteristics. Previous studies have found that different types of muscle fibers can influence meat quality traits, including meat color, tenderness, water-holding capacity, juiciness, and flavor 4,5. In chickens, myofiber can be divided into red and white fibers, which are referred to as oxidative (type I and IIA) and glycolytic fibers (type IIB), respectively. Oxidative fibers exhibit slow contractility and oxidative metabolism based on mitochondrial oxidative phosphorylation, whereas glycolytic fibers have fast contractility and glycolytic metabolism 6,7. Although the differences between various muscle fiber types in physiology and functionality have been well studied, the molecular regulation of their specification and maintenance in chickens remains largely unknown 8,9. miRNAs are highly conserved non-coding small RNAs that regulate gene expression at the post-transcriptional level in most biological processes. Emerging evidence has demonstrated that miRNAs are involved in

ABSTRACT. Chinese black-bone chickens are valued for the medicinal properties of their meat in traditional Chinese medicine. We investigated the genetic diversity and systematic evolution of Chinese black-bone chicken breeds. We sequenced the DNA of 520 bp of the mitochondrial cyt b gene of nine Chinese black-bone chicken breeds, including Silky chicken, Jinhu black-bone chicken, Jiangshan black-bone chicken, Yugan black-bone chicken, Wumeng black-bone chicken, Muchuan black-bone chicken, Xingwen black-bone chicken, Dehua black-bone chicken, and Yanjin black-bone chicken. We found 13 haplotypes. Haplotype and nucleotide diversity of the nine black-bone chicken breeds ranged from 0 to 0.78571 and 0.00081 to 0.00399, respectively. Genetic diversity was the richest in Jinhu black-bone chickens and the lowest in Yanjin black-bone chickens. Analysis of phylogenetic trees for all birds constructed based on hyplotypes indicated that the maternal origin of black-bone chickens is predominantly from three subspecies of red jungle fowl. These results provide basic data useful for protection of black-bone chickens and help determine the origin of domestic chickens.

Back and thigh skin of chickens showed significant differences in the thickness and the feather follicle density and size, which are important traits for slaughtered chickens' appearance. In the present study, global gene expression profiling was conducted in the back and thigh skin of chickens using Microarray technology. The results showed that 676 genes were differentially expressed between back and thigh skin. The expression of the differentially expressed genes (DEGs), including PPP1R3C, IGF1, PTCHD1, HOXB6, FGF9, CAMK4, SHH, BMP8B, FOXN1 and PTGER2, was validated by real‐time quantitative polymerase chain reaction (RT‐qPCR), and the results were consistent with microarray results. Functional analysis revealed that the DEGs were significantly involved in cell proliferation, differentiation, apoptosis, adhesion and transport process, and the pathways were significantly mapped into the ECM‐receptor interaction, peroxisome, focal adhesion, Hedgehog and PPAR signalling pathways. Protein–protein interaction network analysis suggested that signalling pathways related to feathers morphogenesis and development, such as Wnt, FGF, MAPK, SHH and BMP signalling pathways, occupied important positions in the network. Genes involved in these signalling pathways and adhesion molecules might play a vital role in skin and feather follicle development. Further single nucleotide polymorphism (SNP) association analysis of Wnt3A showed that the AC genotype of SNP g.255361 C>A significantly increased the feather follicle density of thigh skin. Our findings may provide new insights on candidate genes and pathways related to skin and feather follicle formation of chickens.

During the early incubation period of the duck, from embryonic day 1 to 13, a precise identification of the sex may be difficult. In a preliminary test, we found a defect in the use of the classical P2/P8, 1237L/1272H, and 2550F/2718R primers for chromo-helicase-DNA-binding 1 gene (CHD1) as a PCR-based test to identify sex in ducks. Therefore, universal PCR primers HPF/HPR for sexing ducks were designed. The PCR product was cloned, sequenced, and analyzed using GenBank. The effectiveness of the primers was compared using samples of blood and feathers from adult birds and chorioallantoic membranes and allantoic fluid (AF) of embryos as a source of DNA. The 495-bp CHD1-Z and the 351-bp CHD1-W PCR amplicons could be easily distinguished on a 3% agarose gel, and females (ZW) displayed 2 visible bands whereas only a single band was found in males (ZZ). The results indicated that HPF/HPR primers were highly efficient and more reliable than the classical primers used for sexing ducks. During the design of the new primers, an AF sampling technique was established to collect a very small amount of AF from free-living birds. This technique, which was minimally invasive, had no adverse effects on either embryos or the post-hatching survival of young ducks and could be used in developmental biology research in birds.