【作者】 张跟喜；

【导师】 王金玉；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本研究利用29个微卫星标记研究边鸡0世代群体的遗传多样性,并与2个对照品种(京海黄鸡和尤溪麻鸡)的遗传多样性进行比较;选择0世代边鸡群体中等位基因数在4个以上的21个微卫星标记研究1世代边鸡群体的遗传多样性,并与0世代边鸡群体的遗传多样性进行比较以了解边鸡群体的保种效果;采用PCR-SSCP技术检测边鸡及3个对照品种(京海黄鸡、尤溪麻鸡和AA鸡)肌肉生长抑制素基因(Myostatin)的单核苷酸多态性,并与边鸡的生长和繁殖性状进行相关分析,为边鸡群体的标记辅助选择提供依据;运用荧光定量PCR技术分析边鸡Myostatin基因G2283A位点形成的3种基因型在胸肌、腿肌和卵巢中表达量的差异,以探讨这一位点对边鸡的生长性状和繁殖性状具有显著效应的分子机理,同时分析边鸡Myostatin基因在胸肌、腿肌和卵巢中表达量的差异。主要研究结果如下:1.利用29个微卫星标记在3个鸡品种(边鸡、京海黄鸡和尤溪麻鸡)中总共检测到166个等位基因,其中32个等位基因为某一品种所特有。边鸡拥有的特有等位基因最多,为15个(46.9%),京海黄鸡次之,为12个(37.5%)。边鸡的平均多态信息含量(0.5168)和平均期望杂合度(0.5750)最高,京海黄鸡的平均多态信息含量(0.4915)和平均期望杂合度最低(0.5505)。在29个微卫星位点中,边鸡群体有15位点为高度多态位点,其余14个位点为中度多态位点。京海黄鸡和尤溪麻鸡的高度多态位点数分别为17和14个。在3个品种中一些位点显著的偏离哈代-温伯格平衡,杂合子缺失的水平也很高。通过固定指数(Fst)估计品种间的遗传分化为6.7%(P<0.001)。群体内的杂合子缺失(Fis)为22.2%(P<0.001)。边鸡的近交系数最高(0.249),京海黄鸡的最低(0.159)。这些研究结果可为边鸡、京海黄鸡以及尤溪麻鸡的遗传特征研究和保种提供初步依据。2.选用的等位基因数在4个以上的21个微卫星标记在2个世代的边鸡群体中共检测到122个等位基因,其中102个为两个世代所共有,19个为0世代所特有。0世代和1世代的边鸡群体的平均多态信息含量(PIC)分别为0.5473和0.5437;平均期望杂合度分别(He)为0.5967和0.6009;近交系数(Fis)分别为0.233和0.134。经过一个世代的繁育,PIC和He两个指标维持在原有水平,而群体的近交系数有所下降,说明采用的保种方法很好的保存了边鸡的遗传多样性,同时避免了近交程度过高而导致群体出现近交衰退。3.在4个鸡品种Myostatin基因中总共检测到17个突变,其中10个突变(A326G、C334G、G673A、G985C、G1085A、A1278T、C1346T、G1375A、A1473G和G1491A)位于5′调控区,4个突变(G2100A、G2109A、C2244G和G2283A)位于外显子1,3个突变(C7552T,C7638T和T7661A)位于外显子3。编码区的突变都未导致氨基酸的改变。4.边鸡、京海黄鸡、尤溪麻鸡和AA鸡Myostatin基因中分别有7、9、9和5个位点表现出多态。边鸡MYOE1-2位点为高度多态位点,MYO1和MYOE1-3位点为中度多态位点,其余4个位点为低度多态位点。京海黄鸡、尤溪麻鸡和AA鸡中高度多态位点数分别为1、2、0个,中度多态位点数都为4个。5.通过在线软件预测发现4个鸡品种Myostatin基因5′调控区的突变总共导致10个转录因子结合位点(E2F、CRE-BP、CREB、HFH-2、NKx-2、CdxA、Oct-1、V-Maf、Tst-1和C/EBPα)发生了改变。6.边鸡Myostatin基因5′调控区MYO1、MYO6和MYO7位点以及外显子区MYOE1-2、MYOE1-3位点对边鸡的生长性状具有显著或极显著的影响(P<0.05或P<0.01),推测Myostatin基因可能是影响边鸡生长性状的主效基因或与主效基因相连锁的一个标记。MYOE1-3位点DE和EE基因型的6-18周龄体重都显著或极显著的高于EE基因型(P<0.05或P<0.01),并且具有EE基因型的体重>DE基因型>EE基因型的规律,MYOE1-3位点在边鸡生长性状的标记辅助选择方面具有很大的应用潜力。7.边鸡Myostatin基因5′调控区MYO1、MYO6和MYO7位点对边鸡的开产体重有显著的影响(P<0.05),外显子区MYOE1-2、MYOE1-3、MYOE3-2和MYOE3-3位点对边鸡的开产日龄和300日龄产蛋数具有显著或极显著的影响(P<0.05或P<0.01),推测Myostatin基因可能是影响边鸡繁殖性状的的主效基因或与主效基因相连锁的一个标记。MYOE1-3位点DE基因型的开产日龄显著的早于DD基因型(P<0.05),EE基因型的开产日龄极显著的早于DD基因型(P<0.01);MYOE1-3位点DE基因型的300日龄产蛋数显著的高于DD基因型(P<0.05),EE基因型的300日龄产蛋数极显著的高于DD基因型(P<0.01),MYOE1-3位点在边鸡300日龄产蛋数的标记辅助选择方面具有很大的应用潜力。8.边鸡Myostatin基因7个多态位点两两组合对边鸡的生长性状和繁殖性状的互作效应达到显著(P<0.05)或极显著(P<0.01)的水平。9.通过荧光定量PCR技术发现Myostatin基因G2283A位点形成的3种基因型(DD、DE和EE)在胸肌、腿肌中的表达量存在差异,在卵巢中的表达量没有差异。在胸肌中,野生型(DD)的表达量是突变杂合型(DE)的表达量的1.40倍,是突变纯合型(EE)的4.66倍。在腿肌中,野生型的表达量是突变杂合型的表达量的1.27倍,是突变纯合型的8倍。研究结果还表明Myostatin基因主要在胸肌和腿肌中表达,在卵巢中只有微量表达,胸肌中的表达量是卵巢中的471.1倍,腿肌中表达量是卵巢中的501.5倍。更多还原

【Abstract】 The level of genetic differentiation and genetic structure in Bian chicken of 0 generation and two controlled chicken populations (Jinghai chicken and Youxi chicken) were analysed based on 29 microsatellite markers; In order to see the effect of conservation in Bian chicken, twenty-one microsatellite markers which had 4 alleles in Bian chicken of 0 generation were choosen to analyse the diversity of Bian chicken in 1 generation and compared the diversity with Bian chicken of 0 generation; PCR-SSCP method was used to detect the SNPs of the Myostatin Gene in Bian chicken, using Jinghai chicken, Youxi chicken and Arbor Acre chicken as controlled populations. We analysed its relationship with growth traits and reproductive traits in Bian chicken as well in order to lay the foundation for Marker-assisted Selection (MAS) for Bian chicken; In order to find out the molecule mechanism that the mutation G2283A had significant effect both on growth traits and reproductive traits, FQ-PCR method was used to study the expreesion level in breast muscle, leg muscle and ovary among 3 genotypes which were formed by the mutation G2283A in the Myostatin gene. The expreesion level in breast muscle, leg muscle and ovary in Bian chicken was also studied. The main results are listed as follows:1. Based on 29 microsatellite markers, a total of 166 distinct alleles were observed across the 3 breeds (Bian chicken, Jinghai chicken and Youxi chicken), and 32 of these alleles (19.3%) were unique to only 1 breed. Bian chicken carried the largest number of private alleles at 15 (46.9%), followed by the Jinghai chicken with 12 private alleles (37.5%). The average polymorphism information content (0.5168) and the average expected heterozygote frequency (0.5750) of the Bian chicken were the highest, and those of the Jinghai chicken were 0.4915 and 0.5505, respectively, which were the lowest. Among 29 microsatellite loci, there were 15 highly informative loci in Bian chicken, and the other 14 were reasonably informative loci. The highly informative loci in Jinghai chicken and Youxi chicken were 17 and 14 respectively. Significant deviations from the Hardy-Weinberg equilibrium were observed at several locus-breed combinations, showing a deficit of heterozygotes in many cases. As a whole, genetic differentiation among the breeds estimated by the fixation index (Fst) were at 6.7% (p<0.001). The heterozygote deficit within population (Fis) was 22.2% (p<0.001), with the highest (0.249) in Bian chicken and lowest (0.159) in Youxi chicken. These results serve as an initial step in the plan for genetic characterization and conservation of the chicken genetic resource of Bian, as well as Jinghai and Youxi chickens.2. Twenty-one microsatellite markers which had 4 alleles were choosen to analyse the diversity of Bian chicken in the two generations. The results showed that a total of 122 alleles were detected, of which, 102 were detected in the two generations, and 9 alleles were unique to generation 0. The average polymorphism information content (PIC) of generations 0 and 1 was 0.5473 and 0.5437, respectively, and the average expected heterozygote frequency (He) of the two generations was 0.5967 and 0.6009, respectively. The inbreeding coefficient was 0.233 in generation 0 and 0.134 in generation 1. Through selection and breeding for one generation, the PIC and He were maintained, while the inbreeding coefficient (Fis) was somewhat decreased. It indicated that the conserved method was feasible. The conservation method not only conserved the genetic diversity of Bian chicken, but also avoided inbreeding depression.3. Seventeen mutations were detected in the four chicken breeds. Amongst them, 10 mutations (A326G, C334G, G673A, G985C, G1085A, A1278T, C1346T, G1375A, A1473G and G1491A) were located in the 5′regulatory region of the Myostatin gene and 4 mutations (G2100A,G2109A,C2244G, G2283A) were located in exon 1. The remaining 3 mutations (C7552T, C7638T, T7661A) were located in exon 3 . Mutations in the coding region of exons were synonymous mutations.4. In Bian chicken, Jinghai chicken, Youxi chicken and AA chicken there were 7, 9, 9 and 5 loci displayed polymorphisms in the Myostatin gene respectively. MYOE1-2 in Bian Chicken was a highly informative locus. MYO1 and MYOE1-3 were reasonably informative loci. The remaing 4 were slightly informative loci. The highly informative loci in Jianghai Chicken, Youxi Chicken and AA Chicken were 1, 2 and 0 respectively. The reasonably informative loci were all 4 in Jianghai Chicken, Youxi Chicken and AA Chicken .5. Through online software prediction it showed that the mutations in 5′regulatory region of the Myostatin gene in 4 chicken breeds resulted in 10 transcription factor binding site (E2F, CRE-BP, CREB, HFH-2, NKx-2, CdxA, Oct-1, V-Maf, Tst-1 and C/EBPα) changed.6. MYO1, MYO6 and MYO7 in the 5′regulatory region of the Myostatin gene and MYOE1-2, MYOE1-3 in the exonic region had significant effect on growth traits (P<0.05 or P<0.01). It suggested that Myostatin gene could be the major gene or is linked to the major gene that affects the growth traits in Bian chicken. For primers MYOE1-3, Bian chicken with genotype DE and EE had larger body weight than those with genotype DD (P<0.05 or P<0.01) from 6 to 18 wk of age. MYOE1-3 had a great potential to be used as genetic marker for growth traits in Bian chicken.7. MYO1, MYO6 and MYO7 in the 5′regulatory region of the Myostatin gene had significant effect on body weight at first egg (P<0.05). MYOE1-2, MYOE1-3, MYOE3-2 and MYOE3-3 in the exonic region had significant effect on age at first egg and egg number at 300 days (P<0.05 or P<0.01). It suggested that the Myostatin gene could be the major gene or is linked to the major gene that affects the reproductive traits in Bian chicken. For primers MYOE1-3, Bian chicken with genotype DD had larger age at first egg than those with genotype DE and genotype EE (P<0.05 and P<0.01 respectively). Correspondingly, Bian chicken with genotype EE and DE had larger egg number at 300 days than those with genotype DD (P<0.01 and P<0.05 respectively). MYOE1-3 had a great potential to be used as genetic marker for egg number at 300 days in Bian chicken.8. Significant interaction effect between different loci of the Myostatin gene were found on growth traits and reproductive traits (P<0.05 or P<0.01) in Bian Chicken.9. Using FQ-PCR method, it showed that the expression level was differ in breast muscle and leg muscle among 3 genotypes which were formed by the mutation G2283A in the Myostatin gene, while there was no difference in ovary among the 3 genotypes. In breast muscle, the expression level of genotype DD was 1.40 times than genotype DE and 4.66 times than genotype EE. In leg muscle, the expression level of genotype DD was 1.27 times than genotype DE and 8 times than genotype EE. The results also showed that Myostatin is principally expressed in breast muscle and leg muscle, while the expression level was very low in ovary. The expression level in breast muscle and leg muscle was 471.1 and 501.5 times than that in ovary respectively.更多还原