Molecular Mechanisms Of Distinct Primary Myofiber Formation Time In Different Pig Breeds

Meat production of adult pig is determined by total myofiber number,while during embryonic stages,total myofiber number is determined by primary myofiber number.Thus,primary myofiber is a crucial determinant factor of adult meat production.Previous studies revealed that primary myofiber formation showed up earlier in pigs with less meat production,however,the mechanisms behind the phenomenon is still unclear.Understanding the molecular mechanisms of distinct primary myofiber formation time in pig breeds with different meat production will help to improve pig meat production by molecular genetic methods and benefit the researches concerning human muscular diseases.In this study,we use combined methods of histomorphology,transcriptome,proteome and whole genome DNA methylation sequencing to understand the molecular mechanisms of distinct primary myofiber formation time in pig breeds with different meat production by utilizing longissimus dorsi muscle of three pig breeds with degressive meat production(Landrace,Lantang and Wuzhishan)during 6 early embryonic stages(18~42 dpc):Through conducting histomorphology and myofiber differentiation marker protein MyHC detection using 18,21,28,35,42 dpc longissimus dorsi muscle of three pig breeds,we found that primary myofiber formation time distincted among three pig breeds: Wuzhishan and Lantang pig showed the first onset of primary myofiber at 32 dpc,followed by Landrace at 35 dpc,while at 35 dpc,the other two pig breeds had significantly higher number of primary myofiber.This again proved previous studies that pig breeds with more meat production showed later formation of primary myofiber.We speculate that pig breeds with more meat production tended to have more time for myoblast proliferation,thus they show later and more primary myofibers,leading to more total myofiber number and adult meat production.Gene expression profiles by RNA-seq further validated the above-mentioned study.After the above-mentioned longissimus dorsi muscle samples for RNA-seq,we found that Landrace showed a stage delay during 18~35 dpc in gene expression patterns than other two pig breeds and distinct myogenic gene expression patterns of three pig breeds had appeared before primary myofiber formation.We also found that myogenic genes/miRNAs and muscle differentiation markers expressed earlier in Wuzhishan pig and expressed the latest in Landrace.Moreover,significant changes of epigenetic related genes expression pattern was found in comparison between Landrace and Wuzhishan pig during 28~35 dpc.These dramatic changes happened just within the formation period of primary myofiber,which indicated that prior to primary myofiber formation,distinct epigenetic status existed in different pig breeds,leading to distinct expression time of myogenic genes and finally resulted in different primary myofiber formation time.We discovered distinct myogenic related protein expression profiles between Wuzhishan and Landrace pig during early embryonic stages and different myofiber type formation time existed even in early embryonic stages by iTRAQ proteomic analysis.We constructed protein interaction network regulating myofiber type,and we also found that histone H3.3 distincted significantly between Wuzhishan and Landrace.Thus,we deduced that the distinction of myofiber type formation time in two pig breeds during early embryonic stages may be influenced by histone modification.Utlizing whole genome bisulfite sequencing(WGBS)of 18~28 dpc longissimus dorsi muscle samples before primary myofiber formation in Landrace and Wuzhishan pig,we found that there was a DNA methylation reprogramming progress during 18~28 dpc in both pig breeds and Landrace showed higher methylation level than Wuzhishan pig during three stages.Myogenic genes tended to go through demethylation progress earlier in Wuzhishan pig,leading to earlier expression of myogenic genes and thus resulted in earlier formation of primary myofiber than Landrace.In summary,the mechanism of distinct primary myofiber formation time in pig breeds with different meat production is: DNA demethylation and histone modification landscapes in pig breeds with less meat production tend to be earlier prepared for myogenic gene expression,leading to earlier onset of muscle differentiation and result in earlier formation of primary myofiber.