Characteristics Of Flavor Precursors In Hu Sheep Muscle And Screening Of Key Regulatory Genes

Mutton is widely loved by consumers for its excellent nutrition value and unique flavor.Flavor quality is one of the most important indicators to evaluate the quality of mutton.It is determined by the aromatic compounds formed by the precusors substances through a series of complex reactions in the muscle,and its quality directly affects the purchasing tendency of consumers.Given that enzymes regulate the metabolic pathways of flavor precursors,finding genes and molecular markers that affect the metabolism of precursor substances may give a scientific basis for substantially improving mutton flavor.Thus,this study comprehensively explores the characteristics of flavor precursors in sheep from the perspectives of different breeds,intraspecific variation,and distinct muscle types,and identifies key genes that regulate the metabolism of major flavor precursors.The main results were as follows:1.Screening of differentially abundant flavor precursors and their regulatory genes in the muscles between Hu sheep and DorperIn this study,the difference in flavor precursors and transcriptome between Hu sheep and Dorper with different intramuscular fat(IMF)content were investigated using widely targeted metabolomics and RNA-sequencing technologies.Then,the key genes regulating the metabolism of vital precursors were explored by integrating transcriptome and metabolome.Consequently,594 metabolites were detected in sheep longissimus dorsi,and 76 differentially abundant metabolites(DAMs)were identified between Hu sheep and Dorper.No DAMs were observed between distinct IMF content groups within each breed.A total of 10 lysophospholipids(LP),including four lysophospholipid ethanolamines and six lysophospholipid cholines,were identified as the main differential precursors between Hu sheep and Dorper.Furthermore,the weighted gene coexpression network analysis uncovered three differentially coexpression modules that were significantly associated with the content of differential LP in Dorper.From the three modules,GLB1,PLD3,LPCAT2,DGKE,ACOT7,and CH25H genes were identified as key genes regulating the metabolism of LP.2.Analysis of fat-soluble flavor precursors characteristics of longissimus dorsi in Hu sheepThe lipid profiles of Hu sheep were characterized using the widely targeted lipidomics technique.As a result,a total of 947 lipid molecules were identified from434 Hu sheep,the majority of which were glycerides and glycerophospholipids.The coefficient of variation analysis showed that 80.49%of the lipids had a coefficient of variation above 30%,indicating that there was a high natural variation in Hu sheep population.Pearson correlation revealed that the lipids in the same category tended to have an obvious positive correlation,with triglyceride molecules had a strongly positively correlated with each other(r>0.6),implying that the lipids may participate in the same metabolic pathways and may have a high-level genetic correlation of them.An extreme glyceride/glycerophospholipid content model was established to explore the contribution of the two precursors to mutton flavor.The results showed that glycerol primarily affected the synthesis of esters,glycerophospholipids significantly influenced the synthesis of alcohols,and both lipids simultaneously influenced the synthesis of ketones and aldehydes.Overall,glycerophospholipids had a higher effect on the flavor of Hu sheep than triglycerides.Furthermore,21 TGs were found to have a significant effect on IMF deposition and to be significantly positively correlated with flavor compounds(p<0.05),indicating that raising IMF content can improve mutton flavor by increasing TG levels.3.Heritablility estimation and the key genes identification of fat-soluble flavor precursors of longissimus dorsi in Hu sheepIn this study,genome resequencing was performed on 434 Hu sheep at a depth of6.9x.The heritability of 947 lipid molecules was then assessed using the linear mixing model in ASReml software.The results showed that 51.3%(486/947)lipid molecules were with a broad-sense heritability of more than 0.2,and 141 lipid molecules had a high heritability(h~2>0.45).Metabolome-based genome-wide association study(m GWAS)was conducted using r MVP software.The study found that 3882 single nucleotide polymorphism(SNP)sites were significantly associated with 841 lipids(p<4.37e-07).Among them LRRC4C is related to the metabolism of glycerolphospholipid;the SH2D4A gene can affect IMF deposition by regulating TG metabolism;MBOAT1 genes may control the metabolism of two monogalactosyldiacylglycerol(MGDG)molecules.4.Flavor differences among distinct part muscles of Hu sheep and their regulatory networksThe lipid profile,flavor profile,fatty acid profile,and muscular characteristics of triceps brachii,biceps femoris,longissimus dorsi,neck muscle,and flank muscle of Hu sheep were compared comprehensively through widely targeted lipidomics,volatilomics technologies combined with the detection of IMF content and muscle histology.Consequently,the longissimus dorsi and flank muscles showed the highest IMF content,the least amount of muscle fibers,and the smallest muscle fiber diameter,implying that these two parts are more tender than the other three.The changes in total saturated fatty acids(SFA)and monounsaturated fatty acids(MUFA)were completely consistent with IMF content,suggesting that SFA and MUFA play a decisive role in IMF deposition.GC-IMS analysis showed the flavor of triceps brachii,biceps femoris,and longissimus dorsi was more intense than that of neck muscle and flank muscle.Lipidomics discovered 161 differentially abundant lipids among the five parts.In the triceps brachii and biceps femoris,the higher abundance of phosphatidylcholine(PC),which contains C18-C22 polyunsaturated fatty acids,may be an important source of flavor.In addition,integrated transcriptome and lipidome data were used to construct a lipid-gene association network with four expression patterns,from which eight genes(PLD1,PLA2G6,CEPT1,PLPP3,ETNK1,ACOX3,AGPAT1,MBOAT1)regulating PC metabolism were identified.In conclusion,this study used metabolomics and lipidomics techniques to analyze the characteristics of the flavor precusors in sheep muscle under different breeds,intra-breed variation,and different muscle types,and a number of candidate genes regulating the metabolism of important flavor precursors were screened combined with transcriptomic and genomic data.These results may provide a basis for understanding the differences in the flavor of mutton,and provide new ideas for the breeding the new mutton sheep varieties.