Characteristics Of Intramuscular Fat Content And Screening Of Related Molecular Markers In Hu Sheep

In recent years,with the significant improvement of living standards,people have higher requirements for the quality of mutton.On the one hand,it is reflected in the meat color,marble pattern of the mutton and other sensory indicators;on the other hand,it is reflected in the tenderness and juiciness.IMF content is obviously related to the sensory quality and taste quality of mutton,which directly affects the flavor,juiciness,tenderness and meat color.The low content of IMF seriously affects the flavor and mouthfeel of the meat.Compared with sheep’s growth rate and slaughter performance,the research on IMF database and molecular markers is relatively lagging.This thesis spent two years in four batches with the same nutritional level of the total mixed ratio pellet diet to raise 921 Hu sheep weaned male lambs in a single pen until they were 180 days old and then slaughtered.IMF was measured on the longissimus dorsi.At the same time,using the candidate gene method was applied to analyze the expression profiles of FASN,FABP4 and LIPE in different tissues and longissimus dorsi with different IMF,and SNPs within them using DNA mixed pool sequencing combined with improved Multiple Ligase Detection Reaction(i MLDR)high-throughput typing method to perform SNP screening of all exon regions and flanking introns of three genes,and compare them with IMF.The correlation of SNPs and IMF was also investigated,the results were as follow:(1)The IMF content of 921 male Hu sheep lambs was measured by Soxhlet extraction method.The mean value of IMF content in population was 4.01%±1.45%,the maximum value was 11.26%,the minimum value was 1.34%,and the coefficient of variation was 36.16%.Of the 921 Hu sheep measured,345 were slaughtered in summer(August 2018 and August 2019),and average IMF,coefficient of variation,maximum value,minimum value were 4.51%±1.61%,35.60%,11.26% and 1.81%,respectively.The remaining 576 were slaughtered in winter(January 2019 and January 2020),average IMF,coefficient of variation,maximum value,minimum value were3.74%±1.27%,33.87%,9.61%,and 1.34%,respectively.Statistical analysis showed that the IMF content of Hu sheep slaughtered in summer was significantly higher than that in winter(P<0.05),indicating that summer is more conducive for IMF deposition than winter.(2)By referring to relevant literature,FASN,FABP4 and LIPE genes were selected as candidate genes related to IMF deposition,and their m RNA expression characteristics in eleven different tissues of Hu sheep were analyzed by q PCR.Meanwhile,q PCR and Western blot were used to analyze the m RNA and protein expression characteristics in longissimus dorsi with different IMF content.The results showed that FASN gene was expressed in different tissues.The expression level of perirenal lipid was the highest and the lowest in the liver,and the expression level of perirenal lipid was significantly higher than that in other tissues.The expression level of LIPE gene was the highest in perirenal lipid and the lowest in spleen,and the expression level of perirenal lipid was significantly higher than that of longissimus dorsi,heart,spleen,kidney,caudal lipid,mesenteric lipid and subcutaneous adipose(P<0.01),but had no significant difference with liver and testis(P<0.05);FABP4 gene showed the highest expression level in perirenal lipids and the lowest in renal lipids,and the expression level in perirenal lipids was significantly higher than that in other tissues.The variation trend of m RNA abundance and protein abundance of the three genes was the same in different IMF content groups.The expression of FABP4 and FASN genes promoted IMF deposition,while the expression of LIPE gene inhibited IMF deposition.(3)A total of 20 SNPs were detected in LIPE and FASN,while no SNPs was screened in FABP4 gene.Among the 10 SNPs within LIPE,2 SNPs(c.5409T>C and c.6402C>A)were located in the exonic region,of which c.5409T>C(tryptophan/Arginine)is a missense,the other 8 SNPs(c.13A>G,c.1471A>G,c.2126G>A,c.2223C>A,c.4819A>G,c.5124A>G,c.5620G>A and c.6276C>G)were located in the intronic region.Among the 10 SNPs detected within FASN,six of them were located in the exonic region(c.2111C>A,c.9320A>G,c.9325G>A,c.9569G>T,c.11330G>T and c.11606A>G),and c.9320A>G(proline/serine),c.9325G>A(alanine/threonine),c.9569G>T(serine/isoleucine),c.11330G>T(glycine/valine)were missense and four SNPs(c.4666A>G,c.5157A>G,c.9413T>C and c.11621G>A)were located in the intronic region.Subsequently,20 SNPs found in current study were evaluated by i MLDR typing primer design,and 6 SNPs(c.1471A>G,c.6402C>A,c.2111C>A,c.9325G>A,c.9569G>T and c.11330G>T)could not be genotyped by i MLDR.After genotyping and quality control,the genetic parameters of genotyped SNP locus were calculated.Five out of 14 SNPs(c.2126G>A,c.2223C>A,c.5124A>G,c.5157A>G and c.11606A>G)were in high polymorphism(PIC>0.5),2 SNPs(c.4666A>G and c.11621G>A)were in low polymorphism(PIC<0.25),and 7 SNPs(c.13A>G,c.4819A>G,c.5409T>C,c.5620G>A,c.6276C>G,c.9320A>G and c.9413T>C)were in moderate polymorphism(0.25<PIC<0.5).Association analysis found that one SNP(c.4819A>G)in LIPE gene and one SNP(c.9413T>C)in FASN gene were significantly correlated with IMF content(P<0.05).LD analysis identified seven and five haplotypes on LIPE and FASN,respectively,and correlation analysis showed that haplotypes and combination genotypes were not significantly correlated with IMF.