LncEDCH1 g.1703613 T>C regulates chicken carcass traits by targeting miR-196-2-3p

Abstract

Single nucleotide polymorphisms (SNPs) are valuable genetic markers that can provide insights into the genetic diversity and variation within chicken populations. In poultry breeding, SNP analysis is widely utilized to accelerate the selection of desirable traits, improving the efficiency and effectiveness of chicken breeding programs. In our previous research, we identified an association between LncEDCH1 and muscle development. To further investigate its specific mechanism, we conducted SNP detection and performed genotyping, linkage disequilibrium, and haplotype analysis. Our research findings indicate that 16 SNPs in the LncEDCH1. Among these SNPs, g.1703497 C>T and g.1704262 C>T were significantly associated with breast muscle weight percentage, g.1703497 C>T and g.1703613 T>C were significantly associated with leg weight percentage, and g.1703497 C>T, g.1703589 T>C, g.1703613 T>C, g.1703636 C>A, g.1703768 T>C, g.1704079 C>T, g.1704250 T>C, g.1704253 G>A were significantly associated with skin yellowness. Two haplotype blocks composed of 6 SNPs that were significantly associated with wing skin yellowness, breast skin yellowness, full-bore weight, and carcass weight percentage. Furthermore, through dual-luciferase reporter assays, biotin-coupled miRNA pull-down assays, 5-ethynyl-2'-deoxyuridine (EDU) assays, immunofluorescence, and quantitative real-time polymerase chain reaction (qPCR), it has been confirmed that miR-196-2-3p inhibits the expression of LncEDCH1 directly by binding to LncEDCH1 g.1703613T>C, thereby achieving indirect regulation of muscle development. These findings provide valuable molecular markers for chicken molecular breeding and broaden our understanding of the regulatory mechanisms.

Keywords: LncEDCH1; carcass trait; chicken; miR-196-2-3p; single nucleotide polymorphism.

Figure 1

The peak maps of all 3 genotypes of SNPs. SNP1: NC_052556.1: g.1703497 C>T, SNP2: NC_052556.1 g.1703547 C>T, SNP3: NC_052556.1 g.1703589 T>C, SNP4: NC_052556.1 g.1703613 T>C, SNP5: NC_052556.1 g.1703615 T>C, SNP6: NC_052556.1 g.1703636 C>A, SNP7: NC_052556.1 g.1703768 T>C, SNP8: NC_052556.1 g.1703785 T>C, SNP9: NC_052556.1 g.1704079 C>T, SNP10: NC_052556.1 g.1704174 A>G, SNP11: NC_052556.1 g.1704235 A>G, SNP12: NC_052556.1 g.1704250 T>C, SNP13: NC_052556.1 g.1704253 G>A, SNP14: NC_052556.1 g.1704261 G>A, SNP15: NC_052556.1 g.1704262 T>C, SNP16: NC_052556.1 g.1704276 C>T.

Figure 2

Linkage disequilibrium analysis of SNPs of LncEDCH1. (A) LD analysis based on Dʹ. Dʹ = 1 indicates full linkage. (B) LD analysis based on r². r² > 0.33 indicates strong linkage.

Figure 3

Identification of SNP4 of LncEDCH1 as a direct target of miR-193b-3p. (A) The original potential binding site of miR-196-2-3p in LncEDCH1, the mutant sequence resulting from the mutated binding site of miR-196-2-3p in LncEDCH1 (SNP4). (B) The potential interaction model between miR-196-2-3p and LncEDCH1 from RNAhybrid. (C) Luciferase assay was conducted by cotransfecting wild-type or mutant LncEDCH1 SNP4 with miR-196-2-3p mimic or mimic-negative control (NC). (D) The interaction of miR-196-2-3p with LncEDCH1 was determined by biotin-coupled miRNA pull down. (E) The relative expression levels of miR-196-2-3p and LncEDCH1 in miR-196-2-3p overexpression group. (F) The relative expression levels of miR-196-2-3p and LncEDCH1 in miR-196-2-3p inhibition group. (G) The potential interaction model between miR-196-2-3p and MAPK8 from RNAhybrid. (H) The potential interaction model between miR-196-2-3p and MFN2 from RNAhybrid. (I) The expression of LncEDCH1 in chicken muscle varies with different SNP4 alleles. In panels (A-I), results are presented as mean ± SEM and paired t tests were used to assess the statistical significance of differences between means. (**P < 0.01).

Figure 4

miR-196-2-3p inhibits proliferation and promotes differentiation of myoblasts. (A–D) EdU proliferation assay (A), the proliferation rate of myoblast (B), cell cycle analysis (C), and relative mRNA levels of several cell cycle genes (D) with miR-196-2-3p overexpression in CPMs. (E–H) EdU proliferation assay (E), the proliferation rate of myoblast (F), cell cycle analysis (G), and relative mRNA levels of several cell cycle genes (H) with miR-196-2-3p inhibition in CPMs. (I) Relative mRNA expression levels of myoblast differentiation marker genes with miR-196-2-3p overexpression in CPMs. (J) Relative mRNA expression levels of myoblast differentiation marker genes with miR-196-2-3p inhibition in CPMs.