【作者】 张宝；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2008， 硕士

【摘要】 本研究采用PCR-SSCP分子标记和DNA测序技术对ND5、GHSR两个基因在南阳牛、秦川牛、郏县红牛、晋南牛、安格斯牛和中国荷斯坦牛6个群体共714个个体的遗传变异进行分析,并探讨了南阳牛、秦川牛、郏县红牛基因遗传变异与生长性状之间的相关性,为这三个黄牛品种进一步选育从理论上提供帮助。本研究取得以下结论:1.6个黄牛品种ND5基因遗传特性分析应用PCR-SSCP技术对6个黄牛品种mtDNA ND5基因3个基因座进行多态性分析。结果表明:N1与N3基因座的PCR产物经SSCP检测仅存在一种带型。N2基因座存在A、B两种单倍型,发现3个SNP,12900 T>C、12923 A>T、12924 C>T（与NC₀06853对照）,其中,12900 T>C位属于同义突变,12923 A>T、12924 C>T两位突变处于同一密码子的二、三位,致使苯丙氨酸变为酪氨酸。在6个黄牛群体中,N2基因座A单倍型频率分别为0.167、0.134、0.139、0.117、0.041、0.066。2.ND5基因多态性与牛生长性状之间的关联分析ND5基因遗传变异与南阳牛的管围、坐骨端宽、体重、体斜长、体高、日增重等性状之间进行关联分析,发现在6月龄坐骨端宽指标上,B单倍型高于A单倍型,差异极显著（P<0.01）;在6月龄体重、体斜长、体高、日增重等指标上,B单倍型也高于A单倍型,差异显著（P<0.05）。在24月龄体高指标上,A单倍型高于B单倍型,差异极显著（P<0.01）;但在12、18月龄南阳牛群体中没有相关。ND5基因的遗传变异与秦川牛、郏县红牛生长性状间进行关联分析,发现秦川牛与ND5基因变异没有相关。在郏县红牛群体中,坐骨端宽指标是A单倍型高于B单倍型,差异极显著（P<0.01）。3.6个黄牛品种GHSR基因遗传特性分析对GHSR基因全部外显子区共4个基因座G1、G2、G3和G4进行遗传变异分析。首次检测到四个突变SNP,nt-7、nt456、nt667和nt3552（参考XM₅92014的序列,转录起始位点为+1）。G1基因座存在三种带型,在-7位发生了碱基C>A突变。G1基因座等位基因C基因频率在南阳牛、秦川牛、郏县红牛、晋南牛、安格斯牛和中国荷斯坦6个黄牛品种中分别为0.729、0.557、0.543、0.536、0.539、0.852。G2基因座发现两个SNP,分别在456、667位发生了碱基G>A、C>T的突变。该两处突变完全连锁,三种基因型分别命名为AA、AB、BB型,在所有研究群体中,没有检测到BB基因型。G2基因座等位基因A基因频率6个黄牛品种中分别为0.889、0.855、0.891、0.903、0.762、0.910。G4基因座仅发现一个SNP,在3552位发生了碱基C>T突变,存在两种带型,G4基因座等位基因C基因频率在6个黄牛品种中分别为0.556、0.549、0.577、0.540、0.560、0.508。6个群体G1基因座均处于Hardy-Weinberg平衡状态。南阳牛、秦川牛、郏县红牛、晋南牛和安格斯牛G1基因座遗传多态性处于中度多态,荷斯坦牛G1基因座处于低度多态。6个群体G2基因座均处于Hardy-Weinberg平衡状态。郏县红牛和安格斯牛G2基因座遗传多态性处于中度多态,南阳牛、秦川牛、晋南牛和荷斯坦牛G2基因座遗传多态性处于低度多态。其中,荷斯坦牛多态信息含量最低。6个群体G4基因座均处于Hardy-Weinberg不平衡状态。南阳牛、秦川牛、郏县红牛、晋南牛、安格斯牛和荷斯坦牛6个黄牛品种中G4基因座遗传多态性均处于中度多态。4.GHSR基因多态性与生长性状之间的关联分析GHSR基因G1、G2和G4基因座的遗传变异与南阳牛、秦川牛、郏县红牛生长性状之间进行关联分析。G1基因座不同基因型在郏县红牛群体中,BB基因型腰角宽、尻长显著大于AA基因型（P<0.05）;在秦川牛群体中AB基因型腰角宽显著大于BB基因型（P<0.05）。G2基因座南阳牛6月龄体重和日增重AA基因型显著高于AB基因型（P<0.05）,但在12/18/24月龄时差异不显著。郏县红牛和秦川牛群体中,不同基因型与生长性状没有相关。G4基因座不同基因型在南阳牛6月龄指标中,AA基因型坐骨端宽大于AB基因型,达到显著水平（P<0.05）。更多还原

【Abstract】 PCR-SSCP and DNA sequencing techniques was used to detect the genetic diversity of the mtDNA ND5 gene and GHSR gene in Nanyang catlle, Qinchuan cattle, Jiaxian Red cattle, Luxi, Angus, and Chinese Holstein and association analysis were carried out to evaluate the effects of genotypes of candidate genes on growth traits of Nanyang catlle, Qinchuan cattle, and Jiaxian Red cattle. The results showed that:1.The genetic polymorphisms of the mtDNA ND5 gene in six cattle breedsPCR-SSCP and DNA sequencing techniques was used to detect the genetic diversity of the mtDNA ND5 gene. There were only one bandpattern in N1 locus and N3 locus after detected by PCR-SSCP technique. At the N2 locus, two kinds of haplotypes, named A and B, with three SNPs （12900 T >C、12923 A>T、12924 C>T、ref. NC₀06853） were detected. The 12900 T >C SNP is a silent mutation, and 12923 A>T and 12924 C>T SNPs cause Phe to be changed to Tyr. The frequencies of haplotype A in six breeds were 0.167、0.134、0.139、0.117、0.041、0.066, respectively.2.The association between the ND5 gene polymorphisms and the growth traits in three cattle breedsThe correlation analysis between the polymorphism of mtDNA ND5 gene and the body height, body length, body weight, hucklebone width, average day gain and circumference of cannon bone of Nanyang cattle revealed that: at 6 months, haplotype B had greater hucklebone width （P<0.01）, body height, body length, body weight, and average daily gain （P<0.05） than haplotype A. At 24 months, haplotype A had greater body height than haplotype B （P<0.01）. And at 12, 18 months, there was no significant differences between them （P>0.05）.The correlation analysis between the polymorphism of mtDNA ND5 gene and the growth traits of Qinchuan and Jiaxian cattle revealed that: in Qinchuan cattle, there were no significant differences between them （P>0.05）; In Jiaxian cattle population, haplotype A had greater hucklebone width than haplotype B （P<0.01）. 3.The genetic polymorphisms of the GHSR gene in six cattle breedsPCR-SSCP and DNA sequencing techniques was used to detect the genetic diversity of the GHSR gene whole exon with G1, G2, G3 and G.4 loci. And nt-7, nt456, nt 667, nt3552 were detected. （ref. XM₅92014, the start translation site is“+1”.）There were three band patterns in G1 locus, the mutation （C>A） was detected at nt-7 location. The frequencies of Allele C in six breeds were 0.729、0.557、0.543、0.536、0.539、0.852, respectively. There were two SNPs nt456 and nt667 with G>A, C>T were detected in this locus and two bands were found in this locus, named AA and AB. The SNPs at nt456 and nt667 were always in linkage with GC and AT together, respectively; BB genotype was not detected in six cattle breeds. The frequencies of Allele A in six breeds were 0.889、0.855、0.891、0.903、0.762、0.910, respectively. There did not found any mutation in G3 locus. Two bands were detected in G4 locus, the mutation （C>T） was detected at nt3552 location. The frequencies of Allele C in six breeds were 0.556、0.549、0.577、0.540、0.560、0.508, respectively. In this study, G1 locus was in Hardy-Weinberg disequilibrium （P<0.05）. The PIC of detected SNPs in 6 population was moderate polymorphic （0.25<PIC<0.50）, except Holstein was less than 0.25. G2 locus was also in Hardy-Weinberg disequilibrium （P<0.05）. The PIC of detected SNPs in 6 populations was less than 0.25. G4 locus was in Hardy-Weinberg disequilibrium （P>0.05）. The PIC of detected SNPs in 6 population was moderate polymorphic （0.25<PIC<0.50）.4.The association between the GHSR gene polymorphisms and the growth traits in three cattle breedsThe correlation analysis between the polymorphism in G1, G2 and G4 loci of GHSR gene and the growth traits of there cattle breeds revealed that: in the G1 locus, the Hip width and rump length of individuals with genotype BB were significantly higher than that of individuals with genotype AA in Jiaxian population （P<0.05）. The Hip width individuals with genotype AB were significantly higher than that of individuals with genotype BB in Qinchuan population （P<0.05）. In the G2 locus, individual with AA genotype had greater body weight and average daily gain than that of AB genotype in Nanyang cattle at 6 month old.（P<0.05）. But the difference was not exsit in 12/18/24 months Nanyang cattle. Also there were no significant differences between Jiaxian and Qinchuan cattle breeds. In the G4 locus, individual with AA genotype had greater hucklebone width than that of AB genotype in Nanyang cattle at 6 month old （P<0.05）.更多还原