| Molecular marker assisted selection method has gained a certain applicationinsheep breeding,but the object of study mainly concentrated in the ewes multiplets traits.Thisstudy has a relatively slow progress in ram,because there is a smaller number of the ram groups and difficult to obtain reliable data.This study choice four breed sheep total 288 as atest material aimed to find genetic markers associated with semen quality studied these gene polymorphism andits correlation with ram sperm quality and its rich polymorphism gene in production,for theestablishment of a new marker-assisted selection method provides the theory basis.(1)In this study we choose FSHR?LHCGR?MBL2?CD4?ID2?AR?EPPIN?INHA?CYP19?HSP70?AQP7?BMP7?TRIM?CFTR and CAPN gene as candidate genes.Used Sequenon Mass Array to analysis candidate genes polymorphism in four varieties of ram group total of 288 samples.we also research this gene associated with semen quality index such as semen volume,sperm concentration,sperm motility,acrosome intact rate,VAP,VSL,VCL,ALH,BVF,LIN,MAD,STR and scrotum roll length.The main study were as follows:(1)We found five mutation sites from fifteen candidate genes in four varieties of sheep group,this gene names FSHR(rs398233545)?LHCGR(rs398733991)?MBL2(rs401909751)?CD4(rs403107881)?ID2(rs420477086)?(2)FSHR(rs398233545)gene found only one genotype CC in Dorpper sheep.In Suffolk,white Suffolk and poll Dorset sheep there are two kinds of genotype CC and CT.The advantage genotype was CC,and genotype frequency was 0.94,0.96 and 0.9respectively.The dominant of alleles gene frequency of C was 0.97,0.98 and 0.95 respectively.The result of PIC showed that FSHR gene in four varieties of sheep were low polymorphic.Correlation analysis with sheep semen quality showed that the loci of two genoytypes effect on four varieties of sheep traits were no significant(P > 0.05).(3)LHCGR(rs398733991)gene found only one genotype CC in Suffolk sheep.In Dorpper and white Suffolk sheep there are two kinds of genetype CC and CT.There are three genetype CC,CT and TT in poll Dorset sheep.The advantage of genetype was CC and genetype frequency was 0.97,0.89 and 0.9 respectively in four varieties of sheep.The dominant of alleles gene frequency of C was 0.98,0.95 and 0.93 respectively.The results of PIC showed that LHCGR gene in four varieties of sheep were moderately polymorphic.Correlation analysis with sheep semen quality showed that CT type individual of BCF is significantly higher than CC(0.01<P<0.05),CC type individual LIN and STR traits is significantly higher than CT(0.01<P<0.05)and CT type individual MAD traits is significantly higher than CC(P < 0.01).This gene loci in other three breeds had no significant correlation.(4)MBL2(rs401909751)gene found only one genotype GG in Poll Dorset sheep.In Dorpper,Suffolk and White Suffolk groups there are two kinds of genetypes GG and AA.The dominant genetype in Dorpper and White Suffolk was GG and genetype frequency was 0.98 and0.82 respectively.The advantage alleles gene frequency of G was 0.99 and 0.88.The dominant genetype in Suffolk was AA and genetype frequency was 0.62,moreover the advantage alleles gene frequency was 0.59.The results showed that MBL2 gene in Dorpper and White Suffolk groups were low polymorphic and in Suffolk sheep was moderately polymorphic..Correlation analysis with sheep semen quality showed that in Suffolk group AA types individual of volume semen is significantly higher than GG(0.01<P<0.05)and in White Suffolk group AA types individual of BCR and MAD traits is significantly higher than GG.this gene loci in other two breeds had no significant correlation.(5)CD4(rs403107881)gene were discovered that all constituting three genotypes in four varieties sheep.The genetype was GG,GA and AA.The advantage of genetypes was GG and genetype frequency was 0.96,0.51,0.8 and 0.42 respectively.The dominant alleles gene was G and alleles gene frequency was 0.98,0.73,0.84 and 0.57.The results of PIC showed that CD4 gene in Dorpper sheep groups were low polymorphic and in Suffolk,White Suffolk and poll Dorset sheep was moderately polymorphic.Correlation analysis with sheep semen quality showed that in Suffolk groups AA types individual of scrotum perimeter is significantly higher than AG(0.01 <P< 0.05).In White Suffolk groups GG types individual of LIN,MAD and STR traits is significantly higher than AG,but AG types individual of Sperm concentration is significantly higher than GG.In poll Dorset groups AG types individual of volume semen and sperm concentration is significantly higher than GG type individual.(6)ID2(rs420477086)gene were discovered that all constituting three genotypes in four varieties sheep.The genetypes were CC,CT and TT.In dorpper and Suffolk groups the advantage genetype was CC and the genetypes frequency was 0.44,0.45 respectively,moreover the dominant alleles frequency of C was 0.66,0.71 respectively.In White Suffolk and poll Dorset groups the dominant genetypes was CT and the genetypes frequency was 0.54,0.62 respectively,moreover the advantage alleles gene frequency of T was 0.54,0.5 respectively.The results of PIC showed that the four groups were moderately polymorphic.Correlation analysis with sheep semen quality showed that in dorpper groups CC types individual of sperm concentration is significantly higher than CT(0.01<P <0.05).In White Suffolk groups CT and TT types individual of acrosome integrity rate is significantly higher than CC(0.01<P<0.05).this gene loci in Suffolk and pollDorset groups had no significant correlation.(7)First discovered that two import haploid type C-C-G-G-C and C-C-G-G-T-T in the ram group.(8)Confirm that FSHR(rs398233545)gene and ID2(rs420477086)gene SNP loci haplotype can be used as the ht SNP.(9)Confirmed that C-C-G-G-C haplotype correlation with semen volume decreases and the reducing of the sperm vitality;C-C-G-G-T-T haplotype associated with the lower average of sperm.(2)Use semi-automatic biochemical machine detection seven kinds of important biochemical indicators(Ca,P,Mg,GLU,UREA,AST,ALT)in sheeps serum and compared it with ram semen quality traits,the results shows as follows:(1)Dorpper ram serum biochemical index of Mg concentration was significantly lower than other three varieties.(2)The results of biochemical indicators analysis associated with semen quality index showed that the concentration of serum calcium and LIN,STR has significant negative correlation(r=-0.203,P=0.022<0.05;r=-0.197,P=0.026<0.05)means that the lower levels of serum calcium the higher levels of the STR and LIN.the concentration of Serum phosphorus and MAD has significant negative correlation(r=-0.2020.P=0.022<0.05),means that the higher leverls Serum phosphorus the lower levels of the MAD.The concentration of serum magnesium and sperm motility has significant negative correlation(r=-0.234,P=0.004<0.01),means that lower levels of Serum magnesium the higher levels of the sperm motility.The concentration of serum magnesium and VAP,ALH has significant positive correlation.he concentration of serum AST and sperm motility has significant negative correlation(r=-0.21,P=0.009<0.01).The concentration of glucose,urea and ALT in serum of semen quality indicators had no significant correlation.In conclusion,FSHR(rs398233545)gene polymorphism has no correlation with the ram semen quality traits,LHCGR(rs398733991)? MBL2(rs401909751)? CD4(rs403107881)?ID2(rs420477086)polymorphism has dominant correlation with ram semen quality can be used as a molecular marker assisted breeding of candidate genes.When we analysis of the correlation of gene with semen quality also need considering the influence of the biochemical. |
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