MyoD基因对肉牛胴体性状影响的分析

用PCR技术克隆到MyoD基因的第二内含子, 采用PCR-SSCP方法研究了3个黄牛品种(鲁西牛、晋喃牛、秦川牛)及4个杂交肉牛(夏洛莱×鲁西牛、安格斯×鲁西牛、利木赞×鲁西牛、西门塔尔×鲁西牛)群体MyoD基因的多态性, 并分析了基因位点多态性与肉牛肉质性状的相关性。实验结果,在国内首次扩增出肉牛MyoD基因的第二内含子的全部序列, 共261 bp。用SSCP方法检测到MyoD基因内含子2有A和B两个等位基因。测序结果表明该座位的多态性是由于内含子二39 bp处C-T的突变和112 bp处C→G的突变造成的。等位基因B在中国地方品种的分布频率高于引进品种的杂交牛群体。c2检验的结果表明, 在该位点的除夏洛莱和安格斯杂交牛外, 其余五个群体(晋南、鲁西、秦川、西门塔尔杂交牛和利木赞杂交牛)均处于Hardy-Weinberg不平衡状态(P>0.05)。实验群体不同基因型与肉牛的宰前活重、胴体重、净肉重、高档肉重、眼肌面积等性状的影响差异极显著或显著(P<0.01或P<0.05), 并且AA型个体均高于AB型个体。     

线粒体基因组D-Loop区基因多态性与2型糖尿病的相关性

随机选取199例浙江地区2型糖尿病患者与102例正常对照, 采用聚合酶链反应(Polymerase chain re-action, PCR)、基因片段直接测序来检测线粒体基因组D-Loop区域基因变异情况, 同时分析其与主要临床指标的关系。结果显示: 线粒体基因组D-Loop区域为一高变异区, np73A-G、np263A-G、np16223C-T、np16519T-C为4个高变异位点; 发现29个未见报道的新变异位点; np193A-G、np234A-G、np16108C-T等变异与糖尿病家族史有关。这表明浙江籍汉族人线粒体基因组D-Loop区存在大量基因多态性现象, 此区域的某些变异可能与糖尿病的发生发展等具有一定相关性。     

PON基因簇序列变异筛查研究

摘要：系统筛查PON1、PON2及PON3基因编码、剪接及侧翼序列,以期发现所有潜在功能多态基因座,为进一步探讨PON基因家族与心血管疾病的关系做准备。随机选择48例冠心病患者作为筛查对象, 以PCR产物直接测序检测DNA序列变异。扩增片断涵盖整个外显子, 其两侧部分内含子区域及5’和3’侧翼序列。（1）13.9kb测序范围内共发现31个多态性基因座,均为单核甘酸多态（SNP）,其中17个SNP为首次报道。（2）国人中SNP构成和等位基因频率与高加索人群存在显著差异。（3）一个基因内部两个或多个多态性基因座间存在完全或近乎完全连锁不平衡相当常见。中国汉族人群中PON基因簇多个潜在功能多态基因座的识别及这些基因座间的强连锁不平衡状态,为在国人中探讨PON基因簇与心血管疾病关系提供了重要的基础数据。     

小麦全基因组抗赤霉病QTL关联位点特异性SSR标记的筛选、等位变异及效应解析

赤霉病是小麦主要的流行病害之一。借助标记辅助选择将不同数量性状基因座（quantitative trait loci，QTL）聚合是防治赤霉病有效且环保的方法，可以从源头上控制赤霉病并降低籽粒中毒素含量。抗赤霉病QTL在小麦全基因组均有分布，但除了Fhb1、Fhb2等少数位点有比较可靠的鉴别标记，绝大部分位点缺乏有效的位点特异性鉴别标记。简单重复序列（simple sequence repeat，SSR）标记多态性丰富，可以区分自然群体中不同等位变异，方便用于标记辅助育种。基于此，搜集了不同文献中报道的与赤霉病关联的SSR标记386个，并用这些标记构建全基因组赤霉病抗性QTL一致性图谱，接着对这些关联标记进行拷贝数分析，进而选择位点内的单拷贝SSR标记，将这些单拷贝标记在156个品种组成的自然群体中进行扩增，并与三季大田和三季温室环境下赤霉病抗性进行关联，筛选与赤霉病抗性关联的单拷贝SSR标记，明确这些标记在自然群体中的有效等位变异和效应。结果表明，共8个单拷贝SSR标记至少在两季试验中与表型显著关联(P<0.05)，涉及2B、2D、3B、5A、5B、6A、6D、7A染色体，有5个单拷贝标记位点存在有效等位变异。中国地方品种和日本品种携带更多的有利变异，且有利等位变异数目越多的品种赤霉病抗性越好。研究分析的QTL位点及其关联的单拷贝SSR标记可用于赤霉病抗病育种，有利于提高品种赤霉病抗性水平和育种效率。     

不同猪种IGFBP3基因内含子2、3多态性分析

本研究采用PCR-SSCP技术结合DNA直接测序对滇南小耳猪、大白猪、梅山猪、香猪、海南五指山猪封闭群、近交系、海南黑猪系(临高猪,屯昌猪)等8个猪种的IGFBP3基因内含子1、2、3的SNP多态性进行检测,并分析其与五指山猪3个生长阶段体质量的关联性。结果表明:内含子2、3的PCR片断表现有多态性,且出现3种基因型。随后的测序结果表明,它们都在相应的区域存在着SNP变异;内含子3在地方猪种大多表现BB基因型和以B等位基因为主,尤其是小型猪;内含子2在中外不同猪种的基因型频率和等位基因的频率分布则不存在品种的特异性,在地方猪种和外来猪种的分布规律不明显。群体遗传多态性检测显示8个猪种在这2个位点均处于中度多态(0.25PIC0.5);关联分析显示五指山猪IGFBP-3内含子3 AA基因型和内含子2 DD基因型猪在8月龄、12月龄的生长体重分别与AB、BB基因型和CC、CD基因型个体存在显著性差异(p0.05),但在2月龄时没有此规律。     

牙鲆MyoD基因单核苷酸多态性位点的筛选

运用PCR-SSCP技术研究100尾牙鲆(Paralichthys olivaceus)MyoD基因的单核苷酸多态性(Single nucleotide polymorphisms, SNPs), 并将筛选到的突变位点与牙鲆生长性状进行相关性分析。结果表明, 在外显子1和内含子1上存在3个SNPs, 在外显子1 (MyoD2)基因座发现3种基因型AA、AB和BB, G863A突变, 属于同义突变。在内含子MyoD4基因座检测到DD、FF、CD、CE、DE和DF型个体。利用最小二乘法研究MyoD基因多态性位点对牙鲆生长性状的影响。结果表明, 外显子1的SNPs对生长性状无显著影响(P0.05)。内含子1的SNPs对牙鲆的生长性状影响均显著(P0.05)。研究结果为SNPs位点与牙鲆生长性能关联分析奠定了基础。       

MyoG基因多态性对鸭屠宰性状的影响

为了探究肌细胞生成素(MyoG)基因多态性对屠宰性状的影响,本试验分别选取三穗鸭和兴义鸭各60只为材料,采用PCR法结合直接测序进行SNPs检测。在三穗鸭MyoG基因编码区中共发现1个单核苷酸多态位点(SNP)g.1131CT;2个SNPs在兴义鸭的MyoG基因中被找到,分别位于为g.238GC和g.397CG处。这些位点均属同义突变,并未引起所编码氨基酸改变。SNPs与屠宰性状的关联性分析表明,三穗鸭g.1131CT位点对胸肌率有显著影响,兴义鸭g.238GC位点对胸肌率有影响,g.397CG处位点对腿肌重有显著影响。由此可推测MyoG基因发生突变可引起鸭的部分屠宰性状发生改变。     

H-FABP基因多态性与江苏地方山羊品种IMF含量的关系

目的:探讨H -FABP基因多态性与江苏地方山羊品种肌内脂肪含量之间的相关性.方法:PCR - SSCP标记技术检测2个江苏地方山羊品种H -FABP基因部分外显子和内含子区域的多态性,利用最小二乘法分析其与IMF含量的关系.结果:在H - FABP基因第2外显子区域发现1个多态性位点,分别定义为GG和GC2种基因型.构建的固定效应模型显示,基因型GC的IMF含量含量高于基因型GG,在背最长肌和腿肌中GC基因型个体的IMF含量显著高于GG基因型(P＜0.05).结论:H-FABP基因第2外显子132 bp位点处GC基因型具有使IMF含量提高的遗传效应.     

蛋鸭IP3R2基因多态性与蛋品质关联性分析

为了了解IP3R2基因对蛋鸭蛋品质的遗传效应,本研究以樱桃谷鸭、三穗鸭和兴义鸭为实验材料,采用PCR产物直接测序法对IP3R2基因g.70681～g.71200和g.71864～g.72386区域进行单核苷酸多态性筛查,应用一般线性模型分析多态座位对三穗鸭蛋品质的影响。结果显示,在IP3R2基因中g.70681～g.71200共检测到2个SNPs位点,分别为g.70868 CT和g.70871 CT,2个SNPs位点完全连锁,均位于内含子2,共检测到2种基因型:CC和CT,等位基因C的频率在3个鸭品种中均为优势等位基因,三穗鸭群体中该多态座位为低度多态座位,樱桃谷鸭和兴义鸭群体中均为中度多态座位,樱桃谷鸭群体的杂合度最高、有效等位基因数最大,分别为0.475 8和1.907 7,三穗鸭群体则最低,分别为0.284 8和1.398 2;卡方(χ2)检验发现,该多态座位在樱桃谷鸭群体中基因型分布极显著偏离了Hardy-Weinberg平衡(p0.01),三穗鸭和兴义鸭群体基因型分布均未偏离Hardy-Weinberg平衡(p0.05)。关联性分析表明,IP3R2基因多态座位对蛋黄色泽的影响达到显著性水平(p0.05),CC基因型个体显著高于CT基因型个体(p0.05),其他蛋品质指标在2个基因型间差异未能达到显著水平(p0.05)。IP3R2基因g.70681～g.71200多态座位对鸭的蛋壳品质的影响未达显著水平,但却对蛋黄色泽有显著影响,为进一步研究其功能提供参考。     

猪apoC3基因的遗传变异分析

目的:探究猪apoC3基因多态性,为进一步探讨其对脂肪沉积的影响和表达调控等研究提供依据。方法:选取具有不同脂肪沉积特点的3个猪种(可乐猪、贵州白香猪和大约克猪)构建品种DNA池并进行测序,结合各SNP位点测序峰高比值估算等位基因频率,并利用在线软件对不同基因型的转录结合位点及mRNA二级结构进行预测。结果:在3个猪种的apoC3基因中共发现17个SNPs(2个位于5’侧翼区;1个位于外显子3中,为同义突变;1个位于3’非翻译区,其它13个分别位于3个不同内含子中),其中C813G变异增加了一个转录因子GATA-2结合位点,G2280A变异导致mRNA二级结构最小自由能增加0.4kkal/mol。结论:不同猪种间apoC3基因SNPs位点等位基因频率差异较大,而apoC3基因编码区相对保守。     

鸭脂联素基因单核苷酸多态性检测及群体遗传分析

以昆山麻鸭、樱桃谷鸭、高邮鸭、荆江麻鸭、金定鸭, 山麻鸭、龙白鸭和白羽番鸭8个鸭品种为实验材料, 根据鸭脂联素基因开放阅读框序列设计5对引物, 用PCR-SSCP方法进行单核苷酸多态性分析,并对不同品种群体进行群体遗传学分析。结果发现引物4扩增片段上共存有7个单碱基突变, 第430、457、523处的G-A 、A-G、T-C单碱基突变导致第144、153、175个氨基酸分别由丙氨酸（A）变为苏氨酸（T）、异亮氨酸（I）变为缬氨酸(V)、酪氨酸(Y)变为组氨酸(H); 而C507T, T540C, C576T和C597T 4个单碱基突变为沉默突变。鸭群中表现出AA、AB、AC、BB、BC、CC、DD、DE 8种基因型。8种基因型在8个鸭品种间分布存在极显著的差异(P<0.01)。除金定鸭外, 其他品种均处于Hardy-Weinberg平衡状态。群体遗传分析表明金定鸭的纯合度最高, 高邮鸭最低, 其他各群体的纯合度较相近; 金定鸭为低度多态, 高邮鸭为高度多态, 其他品种为中度多态。表明鸭脂联素基因不同品种中具有丰富的单核苷酸多态性, 可以进一步作为候选基因来分析其与脂肪性状的相关性。     

Origin and domestication history of Peking ducks deltermined through microsatellite and mitochondrial marker analysis

In order to elucidate the domestication history of Peking ducks, 190 blood samples from six Chinese indigenous duck breeds were collected with186 individualsgenotyped by 15 microsatellite markers. Both the F_ST and Nei’s standard genetic distances (D_s) from the microsatellite data indicated high genetic differentiation between Peking duck and other Chinese indigenous breeds. The haplotype network with mtDNA data showed that most of the Peking duck haplotypes were distinctly different from those of other domestic breeds. Although the H01 haplotype was shared by all domesticated duck breeds, Peking ducks displayed 12 specific domestic duck haplotypes, including four similar haplotypes H02, H04, H08 and H22, that formed a single haplogroup (A). Both H02 and H22 haplotypes were also shared by mallard and Peking ducks, indicating that Peking ducks originated from wild mallard ducks.     

中国6个国家级保护鸭种群遗传多样性分析

为探讨国家级保护鸭种群的遗传多样性和遗传分化关系,利用17个微卫星标记,对我国6个国家级保护鸭品种资源的遗传多样性进行了分析,统计了平均遗传杂合度(H)、多态信息含量(PIC)、基因多样度(FST)、平均遗传分化系数(GST)和遗传距离等。结果表明,各鸭品种的杂合度较高,除两个群体表现为显著的杂合子缺失外,其他群体均处于Hard-Weinberg平衡状态。6个鸭种群间平均FST值为17.0%,平均遗传分化系数GST为14.7%,各品种平均杂合度为0.706～0.604,平均多态信息含量(PIC)为0.561～0.663。本研究说明我国6个国家级保护鸭品种资源各品种内和品种间的遗传变异大,遗传多样性丰富。     

Cytochrome b Genetic Diversity and Maternal Origin of Chinese Domestic Donkey

To clarify the origin of Chinese domestic donkeys, we investigated the mitochondrial Cytb gene from 244 animals from 13 native breeds. We found 55 variable sites in the Cytb gene sequence and subsequently defined 58 haplotypes. Analysis of haplotypes in combination with Cytb sequences revealed two mitochondrial origins in Chinese domestic donkeys, phenotypically expressed by the Somalian and Nubian lineages. The Somalian lineage predominated in Chinese domestic donkey breeds. Five specific Cytb gene SNPs diagnostic of each of the lineages were found in this study: 225(T-C), 237(C-T), 915(C-T), 1014(C-T), and 1134(A-G) mutations. They effectively distinguish the Nubian from the Somalian lineage in the mtDNA Cytb gene. Both lineages are from Africa and thus support the African maternal origins of Chinese domestic donkeys. No obvious geographic structure was found in Chinese domestic donkey breeds, but the population showed abundant genetic diversity.     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

Molecular cloning, sequence characterization, SNP detection, and tissue expression analysis of duck FMO3 gene

Flavin-containing monooxygenase 3 (FMO3) is an important monooxygenase for catalytic oxygenation of many harmful xenobiotics. Mutations in the FMO3 gene have been identified as causing trimethylaminuria in human and fishy off-flavor in cow milk and chicken eggs. In this study, the full-length cDNA sequence of Pekin duck FMO3 gene was cloned, sequenced, and characterized. The full-length cDNA sequence consisted of 1,846 bp and contained a 1,599 bp open-reading frame encoding 532 amino acids. Duck FMO3 gene shared a similar nine exon–eight intron structure with chicken and human. The duck FMO3 putative protein sequence showed high identity with that of chicken (82 %), and relative low identity with those of mammals (61–66 %). We also found that the duck FMO3 gene was dramatically expressed in liver, lung, and kidney compared to that in other tissues in the ducks, indicating the possible roles the FMO3 gene could play in the three tissues. By bidirectional sequencing, we also found one nonsense mutation, 5 nonsynonymous, and 21 synonymous mutations in the coding region of the FMO3 gene in 11 duck breeds and some of them were predicted to be potentially associated with the activities of FMO3 protein.     

Genetic Diversity and Systematic Evolution of Chinese Domestic Ducks Along the Yangtze-Huai River

To investigate the population structure and systematic evolution of the domestic duck in China, we sequenced the 667 bp mitochondrial DNA (mtDNA) D-loop control region of 106 ducks from nine breeds along the Yangtze-Huai River. Of the total analyzed sites, 34 (5.1%) were polymorphic due to transitions, transversions, insertions, and deletions. Nucleotide content was 25.6% A, 33.3% C, 15.2% G, and 25.9% T. In total, 31 haplotypes were identified in the target region; of these, the major haplotype was A7, and nine haplotypes were shared by the tested ducks. The haplotype diversity (Hd) and average nucleotide diversity (Pi) were 0.798% and 0.28%, respectively. Hd was highest in the Jingjiang shelduck, followed by the Youxian and Enshi shelducks, and it was lowest in the Wendeng black duck. Nucleotide diversity (Dxy) among the nine breeds ranged from 0.139 to 0.433%, and the Kimura 2-parameter distances were 0.0013-0.0044. Molecular variance indicated that a very high proportion of the insignificant genetic variance was attributable to variations within breeds. Phylogenetic analysis of 31 haplotypes revealed only one distinct maternal lineage in the tested ducks, and no evidence was found of a contribution of the Anas zonorhyncha group B haplotype to the maternal origin of Chinese domestic duck breeds along the Yangtze-Huai.     

Genetic diversity and population structure of 10 Chinese indigenous egg-type duck breeds assessed by microsatellite polymorphism

The genetic structure and diversity of 10 Chinese indigenous egg-type duck breeds were investigated using 29 microsatellite markers. The total number of animals examined were 569, on average 57 animals per breed were selected. The microsatellite marker set analysed provided 177 alleles (mean 6.1 alleles per locus, ranging from 3 to 10). All populations showed high levels of heterozygosity with the lowest estimate of 0.539 for the Jinding ducks, and the highest 0.609 observed for Jingjiang partridge ducks. The global heterozygote deficit across all populations (F _IT) amounted to −0.363. About 10% of the total genetic variability originated from differences among breeds, with all loci contributing significantly. An unrooted consensus tree was constructed using the NeighborNet tree based on the Reynold’s genetic distance. The structure software was used to assess genetic clustering of these egg-type duck breeds. Clustering analysis provided an accurate representation of the current genetic relations among the breeds. An integrated analysis was undertaken to obtain information on the population dynamics in Chinese indigenous egg-type duck breeds, and to better determine the conservation priorities.     

SNP detection and haplotype analysis in partial sequence of MSTN gene in sheep

Polymerase chain reaction (PCR) products of the MSTN gene amplified from sixty sheep of nine Chinese indigenous sheep breeds and one imported sheep breed were sequenced to identify the single-nucleotide polymorphisms (SNPs) in a 378-bp fragment including intron 2 and exon 3 of the MSTN gene. A total of fifteen SNPs (A1937C, T1942G, C1956T, A1972C, A1990G, A2008C, A2011G, C2019T, A2025C, A2027C, T2085G, T2173C, C2198T, C2210T and C2213T) were detected among the sixty sequenced individuals and they were all located in intron 2. Twelve haplotypes were identified from these fifteen SNPs, of which haplotype I (CGTCGCGTCCGCTTT) and VIII (ATCAAAACAATTCCC) were the two major and basic ones with frequencies of 12.25% and 77.80%, respectively. Haplotype VIII was distributed in all sheep breeds and all individuals of the meat or meat-wool type sheep breeds were homozygous with respect to this haplotype. This suggests that haplotype VIII might be related to meat production traits in sheep. Haplotype I was only distributed in the fur, lambskin type and fur-meat type sheep breeds. This suggests that haplotype I may have some relationship with fur traits in sheep.     

SNP variation in ADRB3 gene reflects the breed difference of sheep populations

The β3-adrenergic receptor (ADRB3), a G-protein coupled receptor, plays a major role in energy metabolism and regulation of lipolysis and homeostasis. We detect the single nucleotide polymorphism (SNP) variation in full-length sequence of ovine ADRB3 gene in 12 domestic sheep populations within four types by polymerase chain reaction-single strand conformation polymorphism and sequencing to reveal the breed difference. Twenty-two SNPs, 12 of which in the exon 1 and ten in the intron, were detected, and 12 new exonic and four new intronic SNPs were found. Most SNPs presented in Shanxi Dam Line and least ones in Dorset. The average SNP number in both meat and dual purpose for meat and wool breeds was significantly higher than general and dual purpose breeds for wool and meat. Frequency of each SNP in studied breeds or types was different. The 18C Del and 1617T Ins majorly existed in dual purpose breeds for wool and meat. The 25A Del, 119C>G and 130C>T were mostly found in the meat and dual purpose for meat and wool breeds. The 1764C>A more frequently presented in meat than in other types. The majority of variations came from within the populations as suggested by analysis of molecular variance. Close relationship presented among the Chinese and western breeds, respectively. In conclusion, SNPs of ovine ADRB3 gene can reflect the breed difference and within- and between-population variations, and to a great extent, the breed relationship.