【作者】 张翔宇；

【导师】 吴登俊；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 胴体成分是绵羊育种中一个非常重要的经济性状。与其它复杂的生理性状相比,表型值的测定也相对简单。因此,针对胴体成分及其相关性状的QTL定位已经有了许多报道。综合这些信息将有助于深入理解胴体成分性状的遗传变异机制。本研究对156个影响胴体成分的QTL进行了综合分析。先将原始的QTL进行映射,并用元分析法对映射后的QTL进行分析,产生了12个MQTL。这些MQTL与原始的QTL相比,定位的准确性都得到了提高。然后,对MQTL和绵羊已经定位的候选基因进行关联分析。同时,收集牛的胴体成分候选基因,通过比较基因组图定位到参考图谱中,建立与MQTL的关联。通过文献检索和收集,共检索到13篇文献,对检索到的文献进行QTL的信息收集,共收集到156个影响绵羊胴体成分的QTL,涉及9个品种,共计9127个个体。其中,影响胴体体重(CW)的QTL 12个,影响脂肪性状(FAT)的QTL 50个,影响活体体重(LW)的QTL 33个,影响肌肉性状(MUS)的QTL 61个。经映射分析,将搜集的绵羊胴体成分相关的QTL整合在一张遗传图谱上。映射后的QTL没有覆盖整个基因组,而是分布在1,2,3,4,5,6,8,11,18,20,21号染色体。并且在这些染色体上的QTL分布明显不平衡。2号和18号染色体包括了56%的QTL,2号和18号染色体均包含了25个以上的QTL,而4,8,11和21号染色体上的QTL数目都未超过3个。同时,不同染色体上的分布方式也有所不同,有的表现为簇集在一定区域内。映射后的156个QTL中,有10个是单个QTL,剩下的146个QTL经元分析,产生了12个MQTL。最准确的MQTL分别位于2号和18号染色体上,相应的CI值为4.37 cM、8.98 cM和4.35 cM。MQTL的估计CI值,降低了原始QTL的平均CI值,降低的范围为0.85～72.65 cM。QTL置信区间平均减少40.54 cM,增加了QTL定位的准确性,有利于相关候选基因的鉴定。1号染色体上共有2个元QTL,MQTL1(215.86 cM)和MQTL2(294.37 cM)。2号染色体上共有3个元QTL,MQTL3(95.41 cM)、MQTL4(143.44 cM)和MQTL5(244.58cM)。3号染色体上共有2个元QTL,MQTL6(19 cM)和MQTL7(243 cM)。5号染色体上有1个MQTL8(0.23 cM)。6号染色体上有1个元QTL9(1.06 cM)。18号染色体上共有2个元QTL,MQTL10(76.18 cM)和MQTL11(100.4 cM)。20号染色体上有1个元QTL12(65.26cM)。根据报道的结果,经映射到参考图谱后,有两个已知的绵羊候选基因与MQTL关联。牛基因组中控制胴体成分性状的基因13个,使用虚拟绵羊图谱,3个基因与元QTL存在关联关系。选择与MQTL4关联最紧密的肌肉生成抑制素(MSTN),以及没有与任何MQTL关联的生肌决定因子(MRF)家族作为功能候选基因。采用荧光定量PCR技术,检测了MSTN和MRF家族在出生后绵羊肌肉中的表达方式及表达的组织特异性,以确定这些基因在胴体组成中的具体功能。荧光定量结果表明:肌肉组织中MSTN mRNA的表达量显著高于大脑、心脏和肝脏组织(P＜0.05)。肝脏组织中MSTN mRNA的表达量显著低于肌肉和大脑组织(P＜0.05)。大脑组织和心脏组织间MSTN mRNA的表达量差异不显著。出生后到60日龄背最长肌中MSTN mRNA的表达量不断下降,60日龄时最低,60日龄到105日龄开始上升。105日龄到195日龄保持在一个相对稳定的平台期。之后又开始下降。MRF家族仅在骨骼肌中表达,在非肌肉组织均不表达。在所研究的出生后的肌肉发育过程中,MYOD、MYOG和MYF5在出生不久的骨骼肌中表达量很高,但随年龄的增长,处于不断的减少中,虽然,MYF6 mRNA的表达量105日龄最高,但差异不显著。同时MYF6在研究的整个过程中,表达量没有呈一个固定的变化趋势,基本维持衡定。这一结果表明,MYF6在维持成年肌肉中发挥作用。MYOD、MYOG、MYF5和MYF6的差异表达可能是控制不同肌细胞发育和肌纤维类型的重要方式。同时,采用RT-PCR方法,得到了MYOG和MYF6的CDS全序列。绵羊MYOG基因CDS区核苷酸序列与普通牛之间的同源性为96%。而绵羊MYF6基因CDS区核苷酸序列与普通牛之间的同源性高达99%。本研究筛选出了5个与MQTL关联的候选基因,这些基因可能具有较大的研究价值。结果表明,结合元分析法和比较基因组图是对所研究性状进行新候选基因鉴定的有效方法。另外,MQTL附近的标记有助于标记辅助选择。更多还原

【Abstract】 Carcass composition is one of the most important economical traits in sheep breeding.It is also relatively easy to score when compared to other physiological traits that require heavy experimentation,so that several studies have addressed QTL mapping populations.Synthesizing this information provides a unique opportunity to understand the genetic variation of carcass composition within a large range of diversity.Genetic architecture of carcass composition in sheep was addressed by synthesizing a total of 156 quantitative trait Loci(QTL) available for this trait. These were analyzed first with a projection,and then with a meta-analysis method that yielded a synthetic genetic model with 12 consensus QTL.Meta-analysis led in this case to an increase in the precision in QTL position estimation,when compared to initial QTL position within the corresponding region.The MQTL were compared first to the positions of the few carcass composition candidate genes that have been mapped in sheep.We then projected cattle candidate genes onto the sheep genome using a comparative mapping,analyzed the association of QTL with candidate genes.A total of 156 QTL from 13 studies.Collectively,these studies include data from 9 different breeds and 9127 individual animals.12 were carcass weight(CW),50 were fatness (FAT),33 were live weight(LW),61 were muscle(MUS).A total of 156 QTL have been projected onto the reference map.The QTL for carcass composition are not distributed within the whole sheep genome.All the QTL project to 1,2,3,4,5,6,8,11,18,20 and 21 chromosomes.There was a significant imbalance in QTL breakdown between the 11 chromosomes.Chromosome 2 and 18 together accounted for 56%of QTL.Each 2 and 18 chromosome contained more than 25 QTL, but otherwise chromosome 4,8,11 and 21 contained no more than three QTL.Individual QTL clearly be pooled in one cluster.Of the 156 QTL that could be projected on the reference map,10 were alone in the linkage group,so 146 were candidates for aggregation in the so-called MQTL using a meta-analysis approach.Meta-analysis resulted in 12 MQTL.The most accurate MQTL were located on 2 and 18 chromosomes,with CI values of 4.37 cM,8.98 cM and 4.36 cM, respectively.With respect to the reduction in the length from mean initial to meta-QTL CI,the gain in accuracy ranged from 0.85 cM-72.65 cM.Therefore increases the precision of QTL mapping,which facilitates the identification of relevant candidate genes.Chromosome 1 has been identified two MQTL,MQTL1(215.86 cM) and MQTL2(294.37 cM).Chromosome 2 has been identified three MQTL,MQTL3(95.41 cM),MQTL4(143.44 cM) and MQTL5(244.58 cM). Chromosome 3 has been identified two MQTL,MQTL6(19 cM) and MQTL7(243 cM). Chromosome 5 and Chromosome 6 have been identified only one MQTL,MQTL8(0.23 cM) and QTL9(1.06 cM),respectively.Chromosome 18 has been identified two MQTL,MQTL10(76.18 cM)和MQTL 11(100.4 cM).Chromosome 20 has been identified one MQTL,QTL 12(65.26 cM). Loci involved in carcass composition defined by the meta-analysis method are associated with only two sheep genes already know.We then projected 13 cattle candidate genes onto "Virtual Sheep Genome".This yielded 3 associations between sheep QTL and genes involved in carcass composition in cattle.The Myostatin(MSTN) which was associated the MQTL4 and the myogenic regulator factors(MRFs) which were not associated the MQTL were selected as candidate genes. To investigate the muscle developmental and organizational expression patterns of Myostatin (MSTN),myogenic determining factor(MYOD),myogenin(MYOG),myogenic factor5(MYF5) and myogenic factor5(MYF5),Real-time fluorescence quantitative PCR was applied.Understanding how these factors function requires a detailed expression of these factors in postnatal sheep muscle. Results showed that:the expression level of MSTN mRNA in skeletal muscle was significantly higher than cerebrum,heart and liver(P<0.05).The expression level of MSTN mRNA in liver was significantly lower than cerebrum and skeletal muscle(P<0.05).The expression level of MSTN mRNA in cerebrum and heart were no significantly difference.After birth,MSTN mRNA abundance gradually decreased to its lowest level of expression when sheep were 60 days,but returned back to peak between 60 days and 105 days.And then plateaued between 105 days and 195 days.Thereafter,mRNA decreased again.We here report that MYOD,MYOG and MYF5 transcripts are expressed at high levels in the longissimus of newborn lamb and their level of expression continuously declines throughout postnatal life.MYF6 transcript,on the other hand,is present at a constant level through the life span of the study.This result indicated that this level of MYF6 expression is maintained in the adult.Differences in the expression patterns for MYF5, MYOD,MYOG and MYF6 between skeletal muscle development suggest that the relative timing of expression for each muscle regulatory factor may control the distinct phenotypes associated with myotomal myocytes and multinucleate myofibers.And we obtained the corresponding complete CDS sequences in sheep by RT-PCR method.The homology of MYOG and MYF6 nucleotide sequences between Bos Taurus and sheep were 96%and 99%,respectively. These results highlight five possible candidate genes which associated with MQTL for sheep carcass composition.Results suggest that the combination of meta-analysis within a species of interest and comparative genome map can be an efficient strategy for identifying new candidate genes for trait variation.Markers at this MQTL should prove helpful in marker-assisted selection.更多还原