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. Keywords Chicken • MC1R, TYR and ASIP genes • SNP • Plumage Color Chao-wu Yang and Jin-shan Ran contributed equally to this work, and shall share the first author. Yi-ping Liu and Xiao-song Jiang contributed equally to this work, and shall share the correspondence author.

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The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.

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