| Muscle is a highly specialized tissue required for survival, body movement, and serves as major part of human nutrition. Out of three types of body muscles, skeletal muscle formation is a complex/integrated process which originated from paraxial mesoderm and executed by the action of multiple genes through proliferation and differentiation of myoblasts to form myofibers. This study investigated polymorphisms and functions of two candidate genes Gap junction alpha-1(GJA1) and Histone deacetylase 2(HDAC2) which were identified from our previous GWAS results and had close association with skeletal muscle development in chicken.Experiment-1: This study has examined the possible functional roles of HDAC2 and GJA1 genes in skeletal muscle development of chicken. For the study of HDAC2, gene expression was measured by q PCR in breast and thigh muscles during both embryonic(four time points) and post-hatch(five time points) development and in cultures of primary myoblasts during both proliferation and differentiation. The expression of HDAC2 increased significantly across embryonic days in breast and thigh muscles suggesting its role in myoblast hyperplasia. Expression of HDAC2 during myoblast proliferation in vitro declined between 24 h and 48 h when expression of the marker gene paired box 7(PAX7) increased and cell numbers increased throughout 72 h of culture. During induced differentiation of myoblasts to myotubes, the abundance of HDAC2 and the marker gene myogenic differentiation 1(MYOD1), both increased significantly. In another study, three GJA1 si RNAs(si RNA-1, 2 and 3) at 24 h of proliferation showed lower expression in each si RNA where si RNA-1 was comparatively better than others. Therefore morphological observation by si RNA-1 was conducted at 24 h, 48 h, and 72 h of culture and also found its suppression manner in each time. Similar results observed in m RNA expression during differentiation of myoblast at 24 h, 48 h and 72 h of culture by si RNA-1. Taken together, it is suggested that HDAC2 is most likely involved in a suppressive fashion in myoblast proliferation and may play a positive role in myoblast differentiation where GJA1 induced both myoblast proliferation and differentiation.Experiment-2: This study aimed to screen single nucleotide polymorphisms(SNPs) in chicken GJA1 and HDAC2 genes by DNA sequencing, respectively, 269 and 176 chickens for each gene including Chinese indigenous Beijing-You(BJY) and commercial Cobb broiler(CB) strains. HRM(high resolution melting) analysis was performed to genotype one variant of GJA1 gene located at exon. Totally four variants were detected in chicken GJA1 gene, among which one synonymous mutation located at exon(C61223231T or c.-1110C>T), two at intron(A61229799C or c.5460A>C, T61229928 A or c.5589T>A) and one at promoter(A61230599C or c.6260A>C) regions. Two mutations T64230025 C and A64229858 C were identified in the promoter regions of HDAC2 gene. More genetic diversity observed in BJY breed than commercial CB for both genes. Every SNP of GJA1 had significant association with growth traits of chickens but no association observed between the SNPs of HDAC2 and growth traits of chickens based on the association analysis. Strong linkage disequilibrium(LD) observed between C61223231 T and A61229799 C polymorphisms of GJA1 gene, and four undiscovered haplotypes(CA, TC, CC, TA) were constructed from those two mutations. Association analysis between haplotype combinations(diplotype) of GJA1 and growth traits were highly significant where diplotype CC+CC was dominant for all traits. Similarly strong linkage disequilibrium(LD) observed between T64230025 C and A64229858 C polymorphisms of HDAC2 gene but there were no significant differences found from the haplotype combinations of those two variants. We speculated that GJA1 was a major gene affecting chicken growth traits or it was linked with the major gene(s). |
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