Studies On Milk QTLs Gene Polymorphism Of Buffalo

Eight pooled DNA samples of Morra buffalo,Nili-ravi buffalo, Morra-Native crossbreed buffalo,Morra-Nili-Native crossbreed buffalo consisting of high and low milk yield(P＜0.001),and another three control pooled DNA samples of Nili-Native crossbreed buffalo,Guizhou buffalo, Guangxi cattle were prepared in this study.Seven genes including DGAT1-exon8,GH-intron3/exon5,GHR-exon8,PRL-exon3/exon4, PRLR-exon3 are selected as potential locus affecting the milk production traits in buffalo.RFLP(restriction fragment length polymorphism)and SSCP(single strand conformation polymorphism)are used to detect the SNP(single nucleotide polymorphism)sites among these groups.The results show in the following:1)A 411bp fragment of DGAT1-exon8 was cloned and sequenced.The CfrI enzyme digesting results show that these several buffalo groups and Guangxi cattle share the same K(AA dinucleotide)allele genotype.The genotype of Morra buffalo and Guangxi cattle were different from others after assay by SSCP.The sequenced fragments confirmed that there was only one K(AA dinucleotide)allele in the test groups.And these DNA sequences are highly homology(99%)with the cattle sequence reported in the Genbank.The K allelotype may be correlated with the high fat percentage of the buffalo milk directly,and there is a C/T substitution at the 275^thbase in Morra buffalo;2)A 345bp DNA fragment of GH-intron3 was amplified,the SSCP result shows that genotype of Guangxi cattle is different from all other buffalo.The sequenced GH-intron3 fragments resulted 5 nucleotides difference between cattle and buffola.A MspI restriction site were appear(C nucleotide genotype) which have been reported in other papers at this fragment;3)A 404bp gene fragment of GH exon5 was amplified by PCR.The result of SSCP indicates that the genotype of Guangxi cattle is different from buffalo. Two point substitutions were found between buffalo and the bovine sequence, one mutation is found in the 83^rdbase(A→G),which turns the Glutamine to Arginine(Gln→Arg),the other is nonsensical;4)A 165bp gene fragment of GHR gene exon 8 was cloned by PCR.Each group share the the same genotype after assay by SSCP.Sequencing results indicated that there is no difference among the sequencing results of buffalo' and bovines',and the all groups are F allele genotype of F279Y mutation,an new nucleotide mutation was found in the 79^thbase(T→A),which is located in the intron8 of GHR;5)Two gene fragments of the exon3 and exon4 of PRL gene were amplified, which length are 156 bp and 294 bp respectively.The SSCP result shows that both group Guizhou buffalo and Guangxi cattle have a different genotype from the other groups' genotype.The sequenced gene fragment shows that there is no RsaⅠrestriction site being found in other studies.Three nucleotide variation sites have been found between buffalo and cattle,including a new mutation(T→A)in Guizhou buffalo groups,which is a significative mutation, causing amino acid mutations from threonine to serine(T→S);6)A 81 bp gene fragment of PRLR exon3 was amplified,SSCP result shows that both Guizhou buffalo and Guangxi cattle have a different genotype with the other species.The sequencing result reveals that:the sequence is more than 96%identities comparing to that of swine and donkey,and there is a base substitution(C→T)between Guizhou buffalo,Guangxi cattle and the other buffalo species observed,but all groups are the N allele genotype of S18N mutation.