| 395 blood samples from 4 dairy cattle populations, Chinese Holstein, Canadian Holstein, Australia Holstein and Longzhou dairy cattle were collected to extract genomic DNA in this study. The identification and sequence analysis to the bovine acyl-sn-glycerol-3-phosphate acyltransferaseζ(AGPAT6) gene was performed by homology cloning and bioinformatics approach combined with 5' RACE technique. Afterwards, detection of SNPs within some genomic fragments of the AGPAT6 gene and PRLR gene were employed with the use of sequencing and PCR-SSCP, PCR-RFLP method. Finally, the association among AGPAT6 and PRLR gene with milk performance traits of dairy cattle in 4 populations were investigated. The advances have been acquired in 5 points.1. Cloning of bovine AGPAT6 geneThe RACE technology was used to clone bovine AGPAT6 gene 5'-cDNA, and PCR technology was used to clone AGPAT6 gene 8 introns. Exons and introns contained in AGPAT6 genomic DNA were rebuild one sequence that was accepted by GenBank database, its accession number was EF432784.2. Relativity of PCR-SSCP or PCR-RFLP SNPs of the bovine AGPAT6 gene with milk performance traits of dairy cattleIdentification for mutations in the amplified fragments of AGPAT6 gene was performed and the 11 SNPs were detected by PCR-SSCP or PCR-RFLP. These SNPs were genotyped in the experimental populations. The analysis of general lineal model was used to employ to association between the SNPs and milk performance traits of dairy cattle. The results showed as follows:(1) 1 SNP (281T-281C) were detected by PCR-SSCP in intron 2 of AGPAT6 genes. The different genotypes of PCR-SSCP (281T-281C) loci have the significant difference on 305d matured equivalency (305dME), milk fat percentage, and milk protein percentage (P＜0.05).(2) 5 SNPs (410G-410T, 489G-489T, 600G-600T, 649G-649A, 729-729A) were detected by PCR-RFLP in intron 3 of AGPAT6 gene. The different genotypes of PCR-RFLP (410G-410T) loci have the significant difference on milk fat percentage, and somatic cell score (SCS) (P＜0.05). The different genotypes of PCR-RFLP (489G-489T) loci have the significant difference on 305dME, and milk fat percentage (P＜0.05).(3) 2 SNPs (153A-153G, 606A-606G) were detected by PCR-SSCP in intron 4 of AGPAT6 gene. The different genotypes of SNPs loci have the significant difference on SCS (P＜0.05). 3 SNPs (341A-341G, 430C-430T, 451C-451G) were detected by PCR-SSCP in intron 10 of AGPAT6 genes. The different genotypes of PCR-SSCP SNPs loci have the significant difference on SCS (P＜0.05). No SNPs was found in intron 6, 7, 8, 9 of AGPAT6 gene.3. Relativity of PCR-SSCP SNPs of the bovine PRLR gene with milk performance traits of dairy cattleIdentification for mutations in the amplified fragments of PRLR gene was performed and the 11 SNPs were detected by PCR-SSCP. These SNPs were genotyped in the experimental populations. The analysis of general lineal model was used to employ to association between the SNPs and milk performance traits of dairy cattle. The results showed as follows:(1) 3 SNPs (1104A delete, 1267A-1267G, 1268C-1268T) were detected by PCR-SSCP in exon 3 of PRLR gene. The different genotypes of PCR-SSCP SNPs loci have the significant difference on 305dME (P＜0.01).(2) 1 SNPs (3365T-3365C) were detected by PCR-SSCP in exon 5 of PRLR gene. The different genotypes of PCR-SSCP SNPs loci have no significant difference on milk performance traits of dairy cattle.(3) 4 SNPs (5533C-5533T, 5650A-5650G, 5651G-5651A, 5652A-5652T) were detected by PCR-SSCP in exon 7 of PRLR genes. The different genotypes of PCR-SSCP SNPs loci have the significant difference on 305dME, milk fat percentage, milk protein percentage, and SCS(P＜0.05).(4) 1 SNP (7740A-7740G) were detected by PCR-RFLP in intron 8 of PRLR gene. The different genotypes of PCR-RFLP SNP loci have the significant difference on 305dME, and milk protein percentage (P＜0.05).(5) 5 SNPs (9681C-9681T, 9742A-9742G, 9757G-9757T, 9902G-9902C, 9939G insert) were detected by PCR-SSCP in exon 10 of PRLR gene. The different genotypes of PCR-SSCP SNPs loci have the significant difference on 305dME, milk fat percentage, and SCS (P＜0.05).4. Analysis on combination of genotype of 11 SNPs of AGPAT6 gene and genotype of 11 SNPs of PRLR gene effects(1) Combination of genotype of AGPAT6 gene intron 2 and intron 3 with milk performance traits of dairy cattle were analysis. Interaction between genotype of AGPAT6 gene intron 2 and intron 3 were found. The effect of combination 1 (A1A1A2A2C2C2) had significant positive relation with 305dME and milk fat percentage.(2)Combination of genotype of PRLR gene exon 3, 7, 10 and intron 8 with milk performance traits of dairy cattle were analysis. Interaction between genotype of PRLR gene exon 3, 7, 10 and intron 8 were found. The effect of combination 4 (A1A1B3B3A5B5E4E4) had significant positive relation with 305dME.5. Relativity of SNPs of the bovine AGPAT6 and PRLR gene with milk performance traits of Chinese HolsteinIn Chinese Holstein population, 17 SNPs of AGPAT6 and PRLR gene were associated to the milk performance traits of dairy cattle. The result showed as follow:The different genotypes of PCR-SSCP SNPs loci of PRLR gene exon 3 have the significant difference on days of first mate and days of first calving (P＜0.05).The different genotypes of PCR-SSCP SNP loci of PRLR gene exon 5 have the significant difference on milk fat percentage, milk protein percentage, and dry matter of milk content (P＜0.05).The different genotypes of PCR-SSCP SNPs loci of PRLR gene exon 7 have the significant difference on 305dME and days of first calving (P＜0.05).The different genotypes of PCR-SSCP SNPs loci of PRLR gene exon 10 have the significant difference on milk fat percentage (P＜0.05). |
