Screening Of MSTN Gene Mutation Sites That Important For The Development Of Cattle And Sheep Muscles

Cattle and sheep are the world’s main livestock animals and important sources of meat.However,domestic livestock breeds in our country have low meat production,slow growth rate,and long breeding period,among other issues.Therefore,improving the meat production performance of meat-producing livestock is the primary goal of modern breeders.As an important component of animal body weight,muscles not only provide the basis for normal activities of the organism and participate in the regulation of animal energy metabolism but also directly affect the meat production and flavor of livestock.With the development of animal husbandry and the improvement of people’s living standards,cultivating high-yield and high-quality lean livestock with rapid growth has become an increasingly important focus.Therefore,it is crucial to gain a deeper understanding of the variation and function of genes that regulate muscle growth and development.In this study,by exploring the site mutations of MSTN(myostatin,a negative regulatory factor for skeletal muscle development)in different species and studying its tissue-specific regulatory elements,important candidate target sites for gene editing and breeding improvement of ruminant livestock such as cattle and sheep were identified.The main research results are as follows:1.Through collinearity analysis of 27 species,including ruminants and outgroup species,it was found that the functional domains of MSTN and its four receptor genes ACVR2A(activin A receptor type 2A),ACVR2B(activin A receptor type 2B),ACVR1B(activin A receptor type 1B),and TGFBR1(transforming growth factor beta receptor1)is extremely conserved.Two key mutation sites located in the functional domains of MSTN were identified in the subfamily Bovinae: a substitution of lysine(K)to glutamic acid(E)at amino acid position 356,and a substitution of glutamic acid(E)to glycine(G)at position 357.It was also found that the amino acid mutation at position 357 could alter the three-dimensional structure of the MSTN protein,which may affect the binding of MSTN to its type I receptors(ACVR1B and TGFBR1),and subsequently affect downstream pathways.It is tentatively inferred that this may be one of the reasons for the difference in muscle mass between Bovinae and Caprinae animals.2.Through the exploration of intraspecific polymorphic loci in 185 cattle breeds(5116samples),95 goat breeds(510 samples),and 196 sheep breeds(1650 samples),we identified24 missense mutation sites within the MSTN gene interval of the 185 cattle breeds.Similarly,we identified 9 missense mutations within the MSTN gene interval of the 95 goat breeds,as well as 29 missense mutation sites within the 196 sheep breeds.It is worth noting that all these missense mutations are rare,with a frequency lower than 0.01.Additionally,a missense mutation was also discovered in the local breeds of Yanbian cattle and Luxi cattle in China.The discovery of these polymorphic loci provides references for the improvement and breeding of indigenous breeds in our country.3.Through analyzing multi-tissue and multi-stage transcriptome data from cattle,goats,sheep,yaks,and pigs,it was found that MSTN is expressed in various tissues,including muscle,mammary gland,sperm,testis,ovary,brain,spleen,and kidney,and exhibits strong stagespecificity.The expression of MSTN is particularly high in muscle,and it has higher transcriptional activity before birth than after birth,with its expression level corresponding to the growth and development stages of skeletal muscle.The expression of MSTN is extremely high during the formation of primary and secondary muscle fibers,and the number of muscle fibers does not increase after birth,with a significant decrease in the expression of MSTN.4.In order to identify muscle-specific regulatory elements of MSTN,sn ATAC and H3K27 ac data from heart and muscle samples of cattle and mice were analyzed.It was found that MSTN has a higher degree of chromatin openness in muscle,indicating that MSTN has high transcriptional regulatory activity in muscle.A potential key region for regulating MSTN expression was identified in bovine muscle tissue.This finding provides candidate targets for precise regulation of MSTN expression through gene editing and important reference for ruminant breeding improvement.In summary,this study combined multiple omics approaches including comparative genomics,population genetics,transcriptomics,and regulatory genomics to elucidate the genetic variation of MSTN among species and populations,as well as the tissue expression and regulatory specificity of MSTN.These findings provide important potential target sites for gene editing and breeding improvement of ruminant animals such as cattle and sheep.