Studied On Polymorphism Of The 5'-Flanking Region And Exon10 Of Follicle Stimulating Hormone Receptor (FSHR) Gene And Their Relationship To Superovulation In Holstein Cows

In this study,the FSHR gene was chosen as the candidate gene of the breeding trait.Its 5'-regulating region and exon 10 were amplified,blasted and PCR-SSCP tested.The purpose of this study was to find the different sequence of the gene among the different Holstein cows and the relationship of the genotype polymorphism with superovulation in Holstein cows.These studies would be hopeful to find more evidence to optimize the superovulation technology and to increase the number of transferable embryoes at the molecular levels.The results were as follows:1.Two pairs of primers were designed according to the report of the goat FSHR 5' regulating region and the cattle FSHR exon 10 sequence on GenBank. The 5' regulating region and exon10 of FSHR were amplified by the PCR technology,clonned,determined and analyzed with the templates of the DNA sequence of Holstein cows,swamp buffaloes and river buffaloes.The results show that there were differences in the length of FSHR gene 5' regulating region sequence among the three species.Holstein cows,swamp and river buffaloes have 961bp,963bp,and 964bp,respectively.The differences were mainly from the base insertion or deletion.Compared with those in Holstein cows,there were 32 bases mutated,1 base shorted,3 base inserted in swamp buffaloes,and 24 bases mutated,3 bases inserted in river Buffaloes.However,there were 10 bases mutated,1 base shorted between swamp and river buffaloes that the homology between them was 98.9%.The sequence length of exon 10 was the same(1234bp)in the three species. The homology,regarding the exon 10 sequence of FSHR in swamp and river buffaloes compared with that of Holstein cows,was 99.1%and 98.7% respectively,and each has 11 and 15 mutated bases and caused 5 amino acid changes.However,there were 6 bases different and caused 1 amino acid change between swamp and river buffaloes that the homology between them was 99.5%.2.It had been studied by PCR-SSCP to analyze the polymorphism of the 5' regulating region and exon 10 sequence of FSHR gene in Holstein cows.The result shows that 5-1,10-3 and 10-5 fragments were found polymorphism among the ninth sub-sequences of FSHR gene.The polymorphism information content(PIC)values all reached the moderate polymorphism(0.5＞PIC＞0.25).(1)Three genotypes AA,BB and AB were found in the 5-1 segment. Sequence results demonstrated that BB genotype was mutated on such positions as -692(A→G),-667(C→G),-585(C→T)and -550(C→T)compared with AA genotype;Statistical analysis indicated that the predominant genotype was AA, the genotype frequency was 0.50,and the predominant allele was A,the gene frequency was 0.625;The result of X² test was X²=6.3973,X_0.05²ï¼œX²ï¼œX_0.01² (0.01＜P＜0.05).Therefore,Holstein cow groups were not on the Hardy-Weinberg equilibrium status quo.(2)Three genotypes AA,BB and AB were found in the 10-3 segment. Sequence results demonstrated that BB genotype was mutated on such positions as 1473(C→T),1506(T→C)and 1647(A→G)compared with AA genotype; Statistical analysis indicated that the predominant genotype was AA,the genotype frequency was 0.53,and the predominant allele was A,the gene frequency was 0.708;The result of X² test was X²=0.2005,X²ï¼œX_0.05²(P＞0.05). Therefore,Holstein cow groups were on the Hardy-Weinberg equilibrium status quo.(3)Three genotypes AA,BB and AB were found in the 10-5 segment. Sequence results demonstrated that BB genotype was mutated on such positions as 1974(C→G),2008(C→T)and 2056(A→C)compared with AA genotype; Statistical analysis indicated that the predominant genotype was AA,the genotype frequency was 0.58,and the predominant allele was A,the gene frequency was 0.736;The result of X² test was X²=0.8858,X²ï¼œX_0.05²(P＞0.05). Therefore,Holstein cow groups were on the Hardy-Weinberg equilibrium status quo.3.There was no significant difference in the number of ovulation,the number of transferable embryoes and the rate of transferable embryoes in the three genotypes(P＞0.05);However,there was significant difference in the number of ovulation(P=0.0148,P＜0.05)in different ages of the animals,and there was no significant difference in the number of transferable embryoes and the rate of transferable embryoes in different ages of the animals.Furthermore, There was no significant difference in the number of ovulation,the number of transferable embryoes and the rate of transferable embryoes of the age^* genotype.