Myogenesis is a complex and precisely orchestrated process that is highly regulated by several non-coding RNAs and signal pathways. Circular RNAs (circRNAs) represent a novel subclass of endogenous non-coding RNAs that have been identified in multiple species and tissues and play a vital role in post-transcriptional regulation in eukaryotes, but the precise molecular mechanism of action remains largely unknown. Here, we screened a candidate circRNA derived from the SNX29 gene, termed circSNX29 from our previous circRNAs sequencing data of bovine skeletal muscle, and further characterized its regulation and function during muscle development. The overexpression of circSNX29 facilitated myoblasts differentiation and inhibited cell proliferation. Computational analysis using RNAhybrid showed the potential for circSNX29 to sponge to miR-744 with nine potential binding sites. We tested this via a luciferase screening assay and found that circSNX29 directly interacted with miR-744 and downregulation of miR-744 efficiently reversed the suppression of Wnt5a and CaMKIIδ. Importantly, through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, Fluo-4, AM, cell permeant-calcium ion fluorescent probing, and western blotting assays, we found that overexpression of Wnt5a and circSNX29 activated the non-canonical Wnt5a/Ca2+ pathway. Overall, the evidence generated by our study elucidates the regulatory mechanisms of circSNX29 to function as a sponge for miRNA-744 in bovine primary myoblasts.

Circular RNAs (circRNAs) are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes. However, little is known about potential roles for circRNAs in muscle development. We analyzed circRNA sequencing data of bovine skeletal muscle tissue and found differential expression of circTitin (circTTN) in fetal and adult bovine muscle tissue. We then further studied the role of circTTN in bovine muscle development. Overexpression and inhibition of circTTN together elicited its promoting roles in proliferation and differentiation of bovine primary myoblasts. Mechanistically, circTTN showed interaction with miR-432 by luciferase screening and RNA immunoprecipitation (RIP) assays. Additionally, miR-432 is a regulator of insulin-like growth factor 2 (IGF2), as indicated by luciferase activity, quantitative real-time PCR, and western blotting assays. Increased miR-432 expression inhibited the expression of IGF2, but this effect was remitted by circTTN. Conclusively, our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with miR-432 to activate the IGF2/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway.

The detection method based on the mathematical expectation (ME) strategy is fast and accuracy for low frequency mutation screening in large samples. Previous studies have found that the 14-bp insertion/deletion (indel) variants of the 3′ untranslated region (3′ UTR) within bovine PRNP gene have been characterized with low frequency (≤5%) in global breeds outside China, which has not been determined in Chinese cattle breeds yet. Therefore, this study aimed to identify the 14-bp indel within PRNP gene in 5 major Chinese indigenous cattle breeds and to evaluate its associations with phenotypic traits. It was the first time to use ME strategy to detect low frequency indel polymorphisms and found that minor allele frequency was 0.038 (Qinchuan), 0.033 (Xianan), 0.013 (Nanyang), 0.003 (Jiaxian), and zero (Ji'an), respectively. Compared to the traditional detection method by which the sample was screened one by one, the reaction time by using the ME method was decreased 62.5%, 64.9%, 77.6%, 88.9% and 66.4%, respectively. In addition, the 14-bp indel was significantly associated with the growth traits in 2 cattle breeds, with the body length of Qinchuan cattle as well as the body weight and waistline of Xianan cattle. Our results have uncovered that the method based on ME strategy is rapid, reliable, and cost-effective for detecting the low frequency mutation as well as our findings provide a potential valuable theoretical basis for the marker-assisted selection (MAS) in beef cattle.

Background: Chaka sheep, named after Chaka Salt Lake, are adapted to a harsh, highly saline environment. They are known for their high-grade meat quality and are a valuable genetic resource in China. Furthermore, the Chaka sheep breed has been designated a geographical symbol of agricultural products by the Chinese Ministry of Agriculture. Results: The genomes of 10 Chaka sheep were sequenced using next-generation sequencing, and compared to that of additional Chinese sheep breeds (Mongolian: Bayinbuluke and Tan; Tibetan: Oula sheep) to explore its population structure, genetic diversity and positive selection signatures. Principle component analysis and a neighbor-joining tree indicated that Chaka sheep significantly diverged from Bayinbuluke, Tan, and Oula sheep. Moreover, they were found to have descended from unique ancestors (K = 2 and K = 3) according to the structure analysis. The Chaka sheep genome demonstrated comparable genetic diversity from the other three breeds, as indicated by observed heterozygosity (Ho), expected heterozygosity (He), runs of homozygosity (ROH), linkage disequilibrium (LD) decay. The enrichment analysis revealed that in contrast to Mongolian or Tibetan lineage groups, the genes annotated by specific missense mutations of Chaka sheep were enriched with muscle structure development (GO:0061061) factors including insulin-like growth factor 1 (IGF1), growth differentiation factor 3 (GDF3), histone deacetylase 9 (HDAC9), transforming growth factor beta receptor 2 (TGFBR2), and calpain 3 (CAPN3), among others. A genome-wide scan using Fst and XP-CLR revealed a list of muscle-related genes, including neurofibromin 1 (NF1) and myomesin 1 (MYOM1), under potential selection in Chaka sheep compared with other breeds. Conclusions: The comprehensive genome-wide characterization provided the fundamental footprints for breeding and management of the Chaka sheep and confirmed that they harbor unique genetic resources.