【作者】 贺花；

【导师】 刘小林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 以秦川牛共计215个个体为实验研究对象,运用DNA池快速测序法、PCR-SSCP、PCR-RFLP、CRS-PCR-RFLP和DNA序列分析法筛查和鉴定牛CEBPA和AMPD1基因的SNPs,2个候选基因一共筛查到了7个SNPs,其中CEBPA基因筛查到1个SNP,AMPD1基因筛查到6个SNPs,通过对这些SNPs在秦川牛群体中的遗传结构分析、遗传多态性与体尺和胴体性状的关系的研究,目的在于寻找候选基因与秦川牛体尺和胴体性状相关的分子标记,以期为秦川牛生产性能的改良提供科学依据。本实验得到以下重要研究结果:1.秦川牛CEBPA基因SNP筛查及其与体尺和胴体性状的关联分析采用DNA池快速测序的方法寻找秦川牛CEBPA基因SNP,筛查到1个多态位点g.963T>G,该突变位于基因的编码区。运用PCR-RFLP方法验证并分析该位点在秦川牛群体中的多态性。CEBPA-Sma I基因座该基因座Sma I酶切的结果显示:群体中分布A、B两个等位基因和AA、AB、BB三种基因型,处于中度多态。经过卡方适合性检验,秦川牛群体在该基因座达到Hardy-Weinberg平衡状态(P>0.05)。将该基因座的基因型与体尺、胴体性状进行关联分析,结果表明该基因座与宰前活重和胴体重显著相关(P<0.05),BB为优势基因型,B为优势等位基因,暗示该基因座有可能作为秦川牛胴体性状标记辅助选择的标记之一。2.秦川牛AMPD1基因SNPs筛查及其对体尺和胴体性状的关联分析用DNA池快速测序法筛查AMPD1基因的SNPs,筛查到5个SNPs和1个缺失突变,分别是:g.10792A>C,g.14842A>C,g.16214-16231 TTCCCCTCATACCACGCC,g.19416T>C和g.19421A>G,g.22120 C>G。AMPD1-Pst I和AMPD1-Xho I基因座秦川牛AMPD1-Pst I基因座突变位于外显子5;AMPD1-Xho I基因座突变位于内含子7。运用CRS-PCR-RFLP方法分析这两个基因座在秦川牛群体中的多态性,这2个突变位点分别用限制性内切酶Pst I和Xho I酶切的结果显示:群体中均存在A、C两个等位基因,只表现AA和AC两种基因型,没有出现CC基因型;卡方检验表明群体在这两个基因座均达到Hardy-Weinberg平衡状态(P>0.05);PIC值显示群体处于低度多态范围内,说明该基因基因座保守。关联分析后两种基因型与体尺和胴体指标之间没有达到差异显著水平(P>0.05)。AMPD1-D8缺失基因座秦川牛AMPD1基因18 bp的缺失位于基因第16214 bp~16231 bp之间,突变位于内含子8区域。采用DNA测序和琼脂糖凝胶电泳相结合的方法,对缺失区域进行研究。电泳结果出现3种带型,分别为AA野生型,AB缺失杂合型,BB缺失纯合型,存在两种等位基因A和B,基因型频率分别为0.7163、0.2233和0.0605。卡方分析揭示秦川牛群体处于Hardy-Weinberg不平衡状态。三种基因型与相应的体尺和胴体指标关联,分析结果显示:在宰前活重指标上,AA基因型极显著高于AB基因型;胴体重指标上,AA基因型显著高于AB和BB基因型,提示我们缺失突变影响秦川牛的胴体性状。AMPD1-L11连锁基因座秦川牛AMPD1基因g.19416T>C和g.19421A>G两个突变为连锁突变位点,位于基因的内含子11区域,2个突变仅间隔4 bp。利用PCR-SSCP和DNA测序两种方法检测到秦川牛品种中出现4种单倍型,命名为A (T-A),B( T-G),C (C-A)和D (C-G)。PCR-SSCP和DNA测序结果都相互验证此连锁基因座出现5种基因型,描述为AA (T-A/T-A),BC (T-G/C-A),AC (T-A/C-A),CC (C-A/C-A)和CD (C-A/C-G)。五种基因型与相应的体尺和胴体指标相关联,关联结果揭示:在体斜长指标上,BC基因型与AA和CD基因型之间有显著差异(P<0.05);宰前活重指标上,BC基因型也显著高于CD基因型(P<0.05);BC基因型个体比AA、CC和CD型个体有更高的胴体重(P<0.05);屠宰率指标上,BC基因型同样也显著高于AA基因型(P<0.05);其它的体尺和胴体性状关联后均无显著相关(P>0.05)。因此,AMPD1基因的这个连锁基因座对秦川牛的体尺和胴体性状上有显著影响。AMPD1-I15基因座秦川牛此基因座突变位于内含子15。该突变基因座采用PCR-SSCP鉴定其多态性,307 bp PCR产物经PCR-SSCP检测后呈现CC、CG和GG三种基因型。秦川牛此基因座处于Hardy-Weinberg非平衡状态(P<0.05)。PIC为0.318,多态性较丰富。相关分析结果表明:体斜长指标上,GG基因型个体与CC基因型个体达到了统计学的显著水平(P<0.05);胴体重和屠宰率指标上,GG基因型个体与CC基因型个体达到了统计学的极显著水平(P<0.01),并且整个群体中呈现了GG>CG>CC的趋势。更多还原

【Abstract】 The gene pooling and sequencing,PCR-SSCP, PCR-RFLP, CRS-PCR-RFLP and DNA sequence analysis techniques were applied to scan and identification the genetic variations of candidate gene and their association with body measurement and carcass traits in 215 cattles.As a result,7 SNPs was found on the bovine CEBPA and AMPD1 genes.A new SNP was detected at CEBPA gene.6 SNPs was detected at AMPD1 gene.The association between genetic variations and body measurement and carcass traits were analyzed by using general linear model,as well as population genetic structure and distribution of genotype and allele were calculated by POPGENE software.These research will benefit for find effective molecular markers associated to body measurement and carcass traits,and give scientific theoretical basis to the improvement of Qinchuan cattle performance.The research results were displayed as follows:1. SNP detection of CEBPA gene and their assosiation between body measurement and carcass traits in Qinchuan cattleCCAAT enhancer binding protein A (CEBPA) gene was studied as a candidate gene for the body measurement and carcass traits of bovine. A new SNP g.963T>G was detected by sequencing at CDS region. Different genotypes were determined in Qinchuan cattle by restriction fragment length polymorphism (RFLP).CEBPA-Sma I Locus PCR products digested with Sma I demonstrated polymorphisms in analyzed population.We found two allelic and three genotypes in Qinchuan population.The value of polymorphism information content indicated that this was a moderate polymorphism in Qinchuan cattle. Theχ2 test indicated that the polymorphic locus in Qinchuan cattle did fit Hardy-Weinberg equilibrium (P>0.05). g.963T>G polymorphism significantly associated with carcass weight (P<0.05),genotype BB was a predominant genotype and B was a predominant allele. It is suggest that g.963T>G locus may be one of the marker-assisted selection used as carcass traits in Qinchuan cattle.2. SNPs detection of AMPD1 gene and their assosiation between body measurement and carcass traits in Qinchuan cattleThe AMPD1 was researched in the experiment. 6 SNPs were found.There were 5 SNPs and 1 deletional mutation in AMPD1 gene,namely,g.10792 A>C,g.14842 A>C,Del. g. 16214-16231 TTCCCCTCATACCACGCC, g. 19416T>C and g.19421A>G, g.22120 C>G.AMPD1-Pst I and AMPD1-Xho I Loci A SNP A10792C was detected by sequencing at CDS region. A14842C mutation was detected in intron 7 of AMPD1. Different genotypes were determined in Qinchuan cattle by Creat Restriction enzyme Site PCR (CRS-PCR-RFLP). The PCR products of AMPD1 gene exhibited two genotypes and two alleles.Theχ2 test showed that the genotype distributions in Qinchuan cattle breed was in agreement with Hardy-Weinberg equilibrium (P>0.05). The value of polymorphism information content was less than 0.25,which indicated that this was a low polymorphism in Qinchuan cattle. The association of the A10792C and A14842C mutations of AMPD1 with body measurement and carcass traits of Qinchuan cattle were analyzed,the result revealed that there were no obvious differeces (P>0.05).AMPD1-18 bp deletion Locus The objectives of the present study were focused on detecting deletion mutation in bovine AMPD1 gene, and analyzing its effect on body measurement and carcass traits in Qinchuan cattle by using DNA sequencing and agarose electrophoresis methods. Deletion g.16214-16231 TTCCCCTCATACCACGCC mutation was detected in intron 8 of AMPD1, and was located in 16214bp~16231bp. The 198 bp PCR products of AMPD1 gene exhibited three genotypes and two alleles were revealed: A and B. The frequencies of genotype AA,AB and BB in Qinchuan populations was 0.7163, 0.2233 and 0.0605. Theχ2-test analysis demonstrated that the breed was not in agreement with Hardy-Weinberg equilibrium (P<0.05). The association of the 18 bp deletion mutation of AMPD1 gene with body measurement and carcass traits of Qinchuan cattle were analyzed. The cattle with AA genotype had slaughter weight and carcass weight than those with genotype AB (P<0.01 or P<0.05). These results suggest that the 18 bp deletion mutation may influence the carcass traits in Qinchuan cattle.AMPD1-L11 Locus The linkage site g. 19416T>C and g.19421A>G mutation were detected in intron 11 of AMPD1.The polymorphism of the AMPD1 gene was detected by PCR-SSCP and DNA sequencing methods in 215 individuals from Qinchuan beef cattle breeds. The results showed that four kinds of haplotypes, named: A (T-A), B (T-G), C (C-A) and D (C-G); and the five genotypes were conflated and described as: AA (T-A/T-A), BC (T-G/C-A), AC (T-A/C-A), CC (C-A/C-A) and CD (C-A/C-G). Association of the five genotypes with body measurement and carcass traits in Qinchuan cattle breed.The animals with genotype BC had greater body length compared with genotypes AA (P<0.05) and CD (P<0.05) were observed.The genotypes BC had greater slaughter weight compared with genotypes CD (P<0.05);Individuals with genotype BC was significantly associated with carcass weight (P<0.05),and the genotype BC cattle had greater carcass weight than genotypes AA (P<0.05), CC (P<0.05) and CD (P<0.05), the BC genotype had greater dressing percentage than genotype AA (P<0.05). Other growth and carcass traits in the records had no significant association with genotypes studied. Therefore, the presence of two novel mutations in AMPD1 gene might candidate gene that affects body measurement and carcass traits in Qinchuan cattle.AMPD1-I15 locus The C22120G transversion was detected at intron 15 region.PCR-SSCP was applied to analyze the association of variations of AMPD1 gene with body measurement and carcass traits in Qinchuan cattle.The results showed that 307 bp PCR products were demonstrated three genotypes polymorphism in Qinchuan cattle population,namely,CC,CG and GG,which was not in agreement with Hardy-Weinberg equilibrium (P<0.05). The value of PIC indicated that this was a middle polymorphism in Qinchuan cattle. Correlation analysis revealed that the Individuals with genotype GG had greater body length compared with genotypes CC (P<0.05).The genotypes GG had greater carcass weight and dressing percentage compared with genotypes CC (P<0.01).更多还原