京海黄鸡分子标记与生长及屠宰性状关系的研究
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摘要
本研究以优质肉鸡品种京海黄鸡为试验材料,分析该品种早期生长规律及与早期生长相关的基因在表达上的差异,系统研究E-FABP和Ghrelin基因单核苷酸多态性,以及微卫星位点的遗传多样性,并与该品种的生长及屠宰性状进行联合分析,为京海黄鸡早期生长分子标记辅助选择提供有效的遗传标记。试验测定了京海黄鸡早期生长、屠宰性状,利用3个生长曲线模型、典型性状相关及主成分分析研究了京海黄鸡早期生长规律;利用DDRT-PCR技术分析了3个不同生长阶段京海黄鸡大脑和小肠组织的差异表达情况;利用PCR-SSCP技术研究了E-FABP和Ghrelin基因不同区段的SNPs;采用14对微卫星位点对京海黄鸡的遗传特性进行了探索;并分析了所得SNPs及微卫星多态性与京海黄鸡早期生长及屠宰性状之间的关系。
研究结果如下:
(1)京海黄鸡16周龄公鸡平均体重为1849.57 g,母鸡平均体重为1444.49 g。Logistic、Gompert、Bertalanffy生长曲线模型均能很好的拟合京海黄鸡生长过程,拟合度(R2)均超过了99%,但综合分析表明:Bertalanffy曲线模型最适合。京海黄鸡典型相关分析结果表明:体重性状与体尺性状间相关显著主要是由12周龄及16周龄体重与胫长引起的;体重性状与屠宰性状间相关显著主要反映了12周龄及16周龄体重与胴体重相关密切;体尺性状与屠宰性状间相关显著主要反映出体斜长及胫长与半净膛重相关密切。主成分分析结果表明:前5个主成分能尽可能多的反映屠宰性状原始数据所提供的信息。
(2)初步认为GTPase激活蛋白作为一种激发因子,在雏鸡的生长发育过程中具有重要作用。
(3)E-FABP基因引物P1(P99)在京海黄鸡中检测到AA、CC、AB、AC、BC 5种基因型,BB型缺失,其中A、C等位基因频率均为0.45,B等位基因频率为0.10;引物P95(P98)检测到DD、DE、EE 3种基因型,其中D等位基因频率为0.61,E等位基因频率为0.39。卡方检验表明在这2个基因位点上基因频率和基因型频率都达到了Hardy-Weinberg平衡状态。引物P74检测到Ⅰ、Ⅱ、Ⅲ3种基因型,基因型频率分别为0.71、0.09、0.21。首次在京海黄鸡E-FABP基因研究中检测到6个SNP位点,测序结果表明:引物P1(P99)在第3内含子4269-4288bp处存在“CTTAAATTTTCCTTACATTC”20个碱基缺失;引物P95(P98)在第2内含子4015bp处发生了碱基A→G突变;引物P74在第1内含子2187bp处发生了碱基G→A突变,2268bp处发生了碱基T→C突变,2273bp处发生了碱基T→A突变,2342bp处发生了碱基T→C突变。
(4)Ghrelin基因引物P1在京海黄鸡中检测到AA、BB、CC、AB、BC、AC 6种基因型,A、B、C频率分别为0.36,0.48,0.16;引物P3检测到DD、EE、DE 3种基因型,D、E基因频率分别为:0.81,0.19;这2个基因多态位点都处于Hardy-Weinberg平衡状态。而引物P5只检测到FF、FG 2种基因型,GG型缺失,F、G基因频率分别为0.96,0.04。测序结果发现引物P1扩增产物相对于AA型而言,BB型在DNA序列73bp处存在“TC”2bp缺失,CC型在79bp处存在“CTAACCTG”8bp缺失;引物P3在546bp处发生了碱基T→A突变;引物P5在1274bp处发生了碱基T→C突变。
(5)E-FABP及Ghrelin基因的SNPs多态位点对京海黄鸡的生长及屠宰性状,尤其对体重及屠宰性状有重要影响,可以尝试将其作为相关性状的分子标记用于鸡的分子标记辅助选择。
(6)京海黄鸡试验群各微卫星位点的平均杂合度为0.32;14对微卫星位点的平均多态信息含量为0.59;所有微卫星位点的D值都小于0,说明微卫星位点杂合子少于理论值。京海黄鸡历经7世代选择后其遗传多样性比较丰富,存在继续选择空间。
(7)ADL136、ADL185、MCW0058及MCW0328位点对京海黄鸡早期体重、体尺及屠宰性状、MCW120位点对屠宰性状的影响均达到显著或极显著水平,可尝试运用这5个位点对京海黄鸡进行早期选择、分子标记辅助选择。
In order to give effective molecular markers for the marker-assisted selection (MAS) on early production performance of chickens, the systematic investigation was conducted on Jinghai yellow chicken breed, including the early growth rule and different expression of correlated gene, SNP polymorphism of E-FABP and Ghrelin gene, population genetic diversity, and the relationship between molecular markers and correlation characteristics. The early growth rule was studied with three curve models, canonical correlation analysis and principal component analysis, and the gene differential expression in the brain and small intestine tissues of three growth periods was detected with DDRT-PCR method, the polymorphism and relationship between polymorphism and growth and carcass characteristics were analyzed by PCR-SSCP(PCR-RFLP) and microsatellite marker techniques.
The results were as follows:
(1) The average weight of male and female was 1849.57 g and 1444.49 g respectively at the age of 16 weeks. Logistic, Gompertz and Bertalanffy model can fit the growth very well,and the values of R2 were all exceed 99%, but integrated analysis showed that the Bertalanffy model was the best to this breed. Results by canonical correlation analysis showed that the significant correlation between weight at the age of 16 and 12 weeks and shank length, between weight at the age of 16 and 12 weeks and dressing weight, between body length, shank length and semi-eviserated weight reflected the essential relationship of three characteristics. Results by principal component analysis indicted that the first 5 principal components reflected the information of the carcass characteristic from the original data as much as possible.
(2) The GTPase activating protein played an important role during the growth and development period of chickling as a type of activating factor.
(3) Five genotypes of AA, CC, AB, AC and BC with primer P1 (P99) of E-FABP genes were detected, genotype BB was not found, the allele frequency of A, B and C allele were 0.45, 0.10 and 0.45 respectively. With primer P95 (P98), results showed three genotypes of DD, DE and EE, and the allele frequency of D and E allele was 0.61 and 0.39 respectively. The gene frequencies and genotype frequencies both reached the Hardy-Weinberg equilibrium at those two loci indicated by theχ2 test. With primer P74, three genotypes ofⅠ,ⅡandⅢwere detected, and the allele frequency was 0.71, 0.09, and 0.21 respectively. Through sequencing alignment, results showed that there was“CTTAAATTTTCCTTACATTC”20bp absence on the site of 4269-4288bp with primer P1 (P99) of E-FABP gene, A conversion to G on the site of 4015bp with primer P95 (P98), and G conversion to A on the site of 2187bp, T changed to C on the site of 2268bp and 2342bp respectively, T changed to A on the site of 2273bp with primer P74.
(4) Six genotypes of AA, BB, CC, AB, BC and AC were detected with primer P1 of Ghrelin gene, the allele frequency of A, B and C allele was 0.36, 0.48 and 0.16 respectively; three genotypes of DD, EE and DE were detected with primer P3, the allele frequency of D and E allele was 0.81, 0.19 respectively. The gene frequencies and genotype frequencies were both fit Hardy-Weinberg equilibrium at those two loci. Two genotypes of FF and FG were found with primer P5, and GG genotype was absent. Allele frequency of F and G allele was 0.96 and 0.04 respectively. Through sequencing alignment, for BB genotype, there was“TC”2bp absence on the site of 73bp relative to AA genotype, and for CC genotype,“CTAACCTG”8bp absence on the site of 79bp with primer P1; T changed to A was detected on the site of 546bp with primer P3, and T changed to C on the site of 1274bp with primer P5.
(5) Those SNPs of E-FABP and Ghrelin gene had important effect on the growth and carcass characteristics in Jinghai yellow chicken breed, and could be used to MAS as molecular markers of correlation characteristics.
(6) For Jinghai yellow chicken breed, the average of H (Heterozygosity) and PIC (Polymorphism Information Content) were 0.32 and 0.59 respectively, and the values of D were below 0 among all the loci, which showed that heterozygosity was under theory value. The genetic diversity was comparatively abundant and there was much space for selection though the breed had been bred for 7 generations.
(7) Those loci of ADL136, ADL185, MCW0058, MCW0328 and MCW120 had significant or highly significant effect on the growth and carcass characteristics, which would be used for early selection and MAS in Jinghai yellow chicken breed.
研究结果如下:
(1)京海黄鸡16周龄公鸡平均体重为1849.57 g,母鸡平均体重为1444.49 g。Logistic、Gompert、Bertalanffy生长曲线模型均能很好的拟合京海黄鸡生长过程,拟合度(R2)均超过了99%,但综合分析表明:Bertalanffy曲线模型最适合。京海黄鸡典型相关分析结果表明:体重性状与体尺性状间相关显著主要是由12周龄及16周龄体重与胫长引起的;体重性状与屠宰性状间相关显著主要反映了12周龄及16周龄体重与胴体重相关密切;体尺性状与屠宰性状间相关显著主要反映出体斜长及胫长与半净膛重相关密切。主成分分析结果表明:前5个主成分能尽可能多的反映屠宰性状原始数据所提供的信息。
(2)初步认为GTPase激活蛋白作为一种激发因子,在雏鸡的生长发育过程中具有重要作用。
(3)E-FABP基因引物P1(P99)在京海黄鸡中检测到AA、CC、AB、AC、BC 5种基因型,BB型缺失,其中A、C等位基因频率均为0.45,B等位基因频率为0.10;引物P95(P98)检测到DD、DE、EE 3种基因型,其中D等位基因频率为0.61,E等位基因频率为0.39。卡方检验表明在这2个基因位点上基因频率和基因型频率都达到了Hardy-Weinberg平衡状态。引物P74检测到Ⅰ、Ⅱ、Ⅲ3种基因型,基因型频率分别为0.71、0.09、0.21。首次在京海黄鸡E-FABP基因研究中检测到6个SNP位点,测序结果表明:引物P1(P99)在第3内含子4269-4288bp处存在“CTTAAATTTTCCTTACATTC”20个碱基缺失;引物P95(P98)在第2内含子4015bp处发生了碱基A→G突变;引物P74在第1内含子2187bp处发生了碱基G→A突变,2268bp处发生了碱基T→C突变,2273bp处发生了碱基T→A突变,2342bp处发生了碱基T→C突变。
(4)Ghrelin基因引物P1在京海黄鸡中检测到AA、BB、CC、AB、BC、AC 6种基因型,A、B、C频率分别为0.36,0.48,0.16;引物P3检测到DD、EE、DE 3种基因型,D、E基因频率分别为:0.81,0.19;这2个基因多态位点都处于Hardy-Weinberg平衡状态。而引物P5只检测到FF、FG 2种基因型,GG型缺失,F、G基因频率分别为0.96,0.04。测序结果发现引物P1扩增产物相对于AA型而言,BB型在DNA序列73bp处存在“TC”2bp缺失,CC型在79bp处存在“CTAACCTG”8bp缺失;引物P3在546bp处发生了碱基T→A突变;引物P5在1274bp处发生了碱基T→C突变。
(5)E-FABP及Ghrelin基因的SNPs多态位点对京海黄鸡的生长及屠宰性状,尤其对体重及屠宰性状有重要影响,可以尝试将其作为相关性状的分子标记用于鸡的分子标记辅助选择。
(6)京海黄鸡试验群各微卫星位点的平均杂合度为0.32;14对微卫星位点的平均多态信息含量为0.59;所有微卫星位点的D值都小于0,说明微卫星位点杂合子少于理论值。京海黄鸡历经7世代选择后其遗传多样性比较丰富,存在继续选择空间。
(7)ADL136、ADL185、MCW0058及MCW0328位点对京海黄鸡早期体重、体尺及屠宰性状、MCW120位点对屠宰性状的影响均达到显著或极显著水平,可尝试运用这5个位点对京海黄鸡进行早期选择、分子标记辅助选择。
In order to give effective molecular markers for the marker-assisted selection (MAS) on early production performance of chickens, the systematic investigation was conducted on Jinghai yellow chicken breed, including the early growth rule and different expression of correlated gene, SNP polymorphism of E-FABP and Ghrelin gene, population genetic diversity, and the relationship between molecular markers and correlation characteristics. The early growth rule was studied with three curve models, canonical correlation analysis and principal component analysis, and the gene differential expression in the brain and small intestine tissues of three growth periods was detected with DDRT-PCR method, the polymorphism and relationship between polymorphism and growth and carcass characteristics were analyzed by PCR-SSCP(PCR-RFLP) and microsatellite marker techniques.
The results were as follows:
(1) The average weight of male and female was 1849.57 g and 1444.49 g respectively at the age of 16 weeks. Logistic, Gompertz and Bertalanffy model can fit the growth very well,and the values of R2 were all exceed 99%, but integrated analysis showed that the Bertalanffy model was the best to this breed. Results by canonical correlation analysis showed that the significant correlation between weight at the age of 16 and 12 weeks and shank length, between weight at the age of 16 and 12 weeks and dressing weight, between body length, shank length and semi-eviserated weight reflected the essential relationship of three characteristics. Results by principal component analysis indicted that the first 5 principal components reflected the information of the carcass characteristic from the original data as much as possible.
(2) The GTPase activating protein played an important role during the growth and development period of chickling as a type of activating factor.
(3) Five genotypes of AA, CC, AB, AC and BC with primer P1 (P99) of E-FABP genes were detected, genotype BB was not found, the allele frequency of A, B and C allele were 0.45, 0.10 and 0.45 respectively. With primer P95 (P98), results showed three genotypes of DD, DE and EE, and the allele frequency of D and E allele was 0.61 and 0.39 respectively. The gene frequencies and genotype frequencies both reached the Hardy-Weinberg equilibrium at those two loci indicated by theχ2 test. With primer P74, three genotypes ofⅠ,ⅡandⅢwere detected, and the allele frequency was 0.71, 0.09, and 0.21 respectively. Through sequencing alignment, results showed that there was“CTTAAATTTTCCTTACATTC”20bp absence on the site of 4269-4288bp with primer P1 (P99) of E-FABP gene, A conversion to G on the site of 4015bp with primer P95 (P98), and G conversion to A on the site of 2187bp, T changed to C on the site of 2268bp and 2342bp respectively, T changed to A on the site of 2273bp with primer P74.
(4) Six genotypes of AA, BB, CC, AB, BC and AC were detected with primer P1 of Ghrelin gene, the allele frequency of A, B and C allele was 0.36, 0.48 and 0.16 respectively; three genotypes of DD, EE and DE were detected with primer P3, the allele frequency of D and E allele was 0.81, 0.19 respectively. The gene frequencies and genotype frequencies were both fit Hardy-Weinberg equilibrium at those two loci. Two genotypes of FF and FG were found with primer P5, and GG genotype was absent. Allele frequency of F and G allele was 0.96 and 0.04 respectively. Through sequencing alignment, for BB genotype, there was“TC”2bp absence on the site of 73bp relative to AA genotype, and for CC genotype,“CTAACCTG”8bp absence on the site of 79bp with primer P1; T changed to A was detected on the site of 546bp with primer P3, and T changed to C on the site of 1274bp with primer P5.
(5) Those SNPs of E-FABP and Ghrelin gene had important effect on the growth and carcass characteristics in Jinghai yellow chicken breed, and could be used to MAS as molecular markers of correlation characteristics.
(6) For Jinghai yellow chicken breed, the average of H (Heterozygosity) and PIC (Polymorphism Information Content) were 0.32 and 0.59 respectively, and the values of D were below 0 among all the loci, which showed that heterozygosity was under theory value. The genetic diversity was comparatively abundant and there was much space for selection though the breed had been bred for 7 generations.
(7) Those loci of ADL136, ADL185, MCW0058, MCW0328 and MCW120 had significant or highly significant effect on the growth and carcass characteristics, which would be used for early selection and MAS in Jinghai yellow chicken breed.
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