变性高效液相色谱技术在单核苷酸多态性研究中的应用

人类基因组的单核苷酸多态性(SNPs)研究已成为后基因组时代最重要的内容和目的之一,随之而来的迫切任务是需要适合于自动化且高通量检测SNP的技术。变性高效液相色谱(DHPLC)是近几年发展起来的高效、快速筛检SNP的技术,因其检测SNP的高灵敏度、低成本以及全自动化操作等优点而备受关注。     

绵羊H-FABP基因单核苷酸多态性的研究

以小尾寒羊(48只)、宁夏滩羊(121只)、滩寒杂交羊F1(23只)、无角陶塞特(48只)、萨福克(24只)5个绵羊群体为实验材料, 利用PCR-SSCP和DNA测序技术对心脏型脂肪酸结合蛋白(H-FABP)基因(GenBank登录号: AY157617)外显子2和内含子2部分序列进行单核苷酸多态性(SNPs)检测及遗传多态性分析。结果表明: (1)引物2的PCR扩增产物中存在981(G/A)、1014(A/C)、1019(T/C) 和1058 (-/G ) 4个SNP位点, 表现为AA、BB、CC、AB、AC、BC、AD、CD和BD 9种基因型, 其中AA为优势基因型。经χ2检验后, 除滩羊和萨福克羊外, 其他群体的基因频率和基因型频率均处于Hardy-Weinberg平衡状态。群体遗传多态性分析表明: 宁夏滩羊、小尾寒羊和无角陶塞特羊3个群体中的多态信息含量(PIC)均处于0.25和0.50之间, 为中度多态, 萨福克羊和滩寒杂交羊F1为低度多态(PIC<0.25), 表明脂肪酸结合蛋白基因在不同绵羊品种中具有单核苷酸多态性, 可以进一步作为候选基因来分析其与肌内脂肪含量性状的关联性。(2)引物4的PCR扩增产物中检测到1个SNP多态位点为2407(T/C), 表现为HH、Hh和 hh 3种基因型, 基因型频率大小为HH＞Hh＞hh, 经χ2检验后, 在滩羊和无角陶塞特羊中均为达到Hardy-Weinberg平衡状态, 其多态信息含量均为低度多态(PIC<0.25), 而在小尾寒羊、滩寒杂交羊F1和萨福克羊均没有多态出现。     

我国主要地方绵羊品种随机扩增多态DNA研究

对蒙古羊、湖羊、滩羊、小尾寒羊、乌珠穆沁羊、藏绵羊、阿勒泰羊7个地方绵羊品种和无角陶赛特羊、德国美利奴羊、萨福克羊3个引入品种基因组DNA进行了RAPD分析。结果表明：（1）RAPD可作为一种有效的标记用于绵羊品种之间遗传亲缘关系的分析。（2）在所使用的43种随机引物中,有35种引物扩增出多态谱带,多态频率为66.24%,说明RAPD技术用于研究绵羊核DNA的遗传变异具有较高的检出率和灵敏度。（3）总群体平均遗传多样性指数（HSP)为0.9139,说明绵羊群体具有较为丰富的遗传多样性。（4）我国地方绵羊品种间的分子聚类关系与其所处的地理位置、考古学结果,以及细胞遗传学研究结果基本,引入品种间的分子聚类关系也与其育成史基本一致。     

我国6个地方绵羊品种微卫星DNA多态性研究

利用聚丙烯酰胺凝胶电泳技术研究了我国蒙古羊、乌珠穆沁羊、哈萨克羊、阿勒泰羊、滩羊和藏绵羊 6个地方绵羊品种 17个微卫星标记的多态性 ,以探讨其遗传多样性、起源分化及群体间的遗传亲缘关系。结果表明微卫星标记不同位点间遗传多样性差异极显著 (P <0 0 1) ,群体间多态信息含量 (PIC)、近交程度 (Fis)和观察杂合度 (Obs .Het)差异不显著 ,但基因多样性 (genediversity)和期望杂合度 (Exp .Het)差异显著 (P <0 0 5 )。所研究的我国 6个地方绵羊品种与欧洲品种具有相似的遗传多样性 ,但具有较高的近交系数。个体和群体的聚类分析结果提示我国地方绵羊品种可能起源于两类祖先。群体间的聚类分析结果还表明 ,蒙古羊与乌珠穆沁羊分化不明显且具有较近的遗传亲缘关系 ,蒙古羊与藏绵羊间分化明显且具有较远的遗传亲缘关系。滩羊、阿勒泰羊以及藏绵羊间也具有较近的遗传亲缘关系。所研究的我国 6个地方绵羊品种的遗传分化 (Fst)与西班牙绵羊品种接近 ,但明显小于欧洲其他绵羊品种     

我国主要地方绵羊品种随机扩增多态DNA研究

对蒙古羊、湖羊、滩羊、小尾寒羊、乌珠穆沁羊、藏绵羊、阿勒泰羊7个地方绵羊品种和无角陶赛特羊、德国美利奴羊、萨福克羊3个引入品种基因组DNA进行了RAPD分析。结果表明：（1）RAPD可作为一种有效的标记用于绵羊品种之间遗传亲缘关系的分析。（2）在所使用的43种随机引物中,有35种引物扩增出多态谱带,多态频率为66.24%,说明RAPD技术用于研究绵羊核DNA的遗传变异具有较高的检出率和灵敏度。（3）总群体平均遗传多样性指数（HSP）为0.9139,说明绵羊群体具有较为丰富的遗传多样性。（4）我国地方绵羊品种间的分子聚类关系与其所处的地理位置、考古学结果,以及细胞遗传学研究结果基本一致,引入品种间的分子聚类关系也与其育成史基本一致。 Abstract:The genetic polymorphism and relationship of 7 indigenous sheep breeds of China and 3 imported sheep breeds were studied using random amplified polymorphic DNA (RAPD).The results indicated that the RAPD was an effective marker for the analysis of genetic relationship among sheep breeds.Among 43 arbitrary primers,35 were polymorphic.The percentage of polymorphic markers was 66.24%,which indicated that the RAPD had higher efficiency of polymorphism detection and sensitivity in studying the genetic variation among sheep breeds.The average index of genetic polymorphism for whole population (Hsp) was 0.9139,which showed that the genetic polymorphism was abundant between sheep populations.The genetic relationship between different indigenous sheep breeds in China was in accord with their localities,the results from archeology and cytogenetics and the genetic relationship between imported sheep breeds was in accord with their breeding history.     

肝脂酶基因多态性与冠心病的关系

肝脂酶 (hepaticlipase,HL)对血浆脂蛋白的代谢起重要作用 ,它影响着血浆中高密度脂蛋白 (HDL)的水平以及低密度脂蛋白 (LDL)的种类 ,因此肝脂酶的活性与冠心病的发生具有相关性。肝脂酶基因的单核苷酸多态性 (SNP)与酶的活性相关 ,并影响血浆脂蛋白水平以及冠心病的发生。为研究肝脂酶基因的单核苷酸多态性与中国汉族冠心病的相关性 ,采用聚合酶链反应、变性高效液相色谱及DNA测序等技术对 10 2例经冠状动脉造影确诊的冠心病患者和 84例正常对照的肝脂酶基因 (包括启动子区以及所有外显子 )的SNP进行了研究 ,结果在肝脂酶基因启动子区发现了一未见文献报道的多态位点 ,即- 2T→C转换。经检验 ,对照组和病例组基因型频率的分布符合Hardy Weinberg平衡。冠心病患者组中 - 2C等位基因的携带者 (基因型为TC或CC)显著高于对照组 (5 7.9%versus 4 2 .7% ,χ2 =4 .181,df=2 ,P =0 .0 4 1) ,且冠心病组中 - 2C等位基因的频率显著高于对照组 (χ2 =3.988,df =1,P =0 .0 4 6 ,OR =1.5 8,95 %CI =1.0 1～ 2 .4 7) ;在冠心病组中进一步发现 -2C等位基因与高密度脂蛋白胆固醇水平升高相关 (P <0 .0 5 )。这提示肝脂酶基因的 - 2T→C多态性可能与血浆高密度脂蛋白胆固醇的水平以及冠心病的发生具有相关性。     

猪Mu阿片受体基因单核苷酸多态性分析

Mu阿片受体（简称MOR）属于G蛋白藕联受体,分布在痛觉传导区,以及与情绪和行为有关的区域,影响动物的神经反应和行为表现。该研究以长白猪、大白猪和杜洛克猪为试验材料, 用8对引物对Mu阿片受体基因的5′ UTR区域、整个编码区和3′ UTR区域用PCR-SSCP方法进行了扫描,发现5处突变基因座（GenBank登录号：AF521309）。统计结果发现基因型频率分布与品种有关,大白猪突变基因型频率显著高于长白和杜洛克,本研究推测分布上的差异可能是由于长期的选择压力造成的。 Abstract:Mu opioid receptor (MOR) is a member of G protein-coupled receptor family,distributed in the pain transduction region in the brain and related to emotion and behaviour.This study was designed to investigate the Single Nucleotide Polymorphism (SNP) of Mu opioid receptor gene in various breeds,including duroc,landrace and Yorkshire.5′ UTR ( untranslate region),coding region and 3′ UTR of Mu opioid receptor gene were amplified by eight pairs of primers,and the Single Nucleotide Polymorphism (SNP) were detected by SSCP.Five polymorphisms were found (Genebank Accession number:AF521309).The results of χ2 test showed that the frequencies of genotypes in different breeds were significantly different (P＜0.01).The frequencies of mutation genotypes in Yorkshire were significantly higher than Duroc and Landrace.According to the above results,we can speculate the difference of the frequencies of genotypes may be the results of long term choice pressure.     

鸡瘦蛋白受体(OBR)基因内含子8单核苷酸多态性与体脂性状的相关研究

以东北农业大学高低脂双向选择系的第 6世代肉鸡为材料 ,鸡 7周龄时测定体重和腹脂重等屠体性状。根据鸡瘦蛋白受体基因内含子 8的序列 (GenBank登陆号 :AF2 2 2 783 )设计引物 ,用直接测序的方法检测多态性位点 ,用PCR SSCP的方法进行基因型分析 ,建立适合的统计模型对多态性位点产生的基因型与生长和体组成性状进行相关分析。结果表明 ,在第 50 0和 659位碱基同时发生了T—C、G—A突变。经最小二乘分析 ,3种基因型在腹脂重和腹脂率上差异显著 (P <0 0 5) ,BB型个体腹脂重和腹脂率显著高于AB型 (P <0 0 5) ,极显著地高于AA型个体 (P <0 0 1) ;3种基因型在肝重上差异显著 (P <0 0 5) ,且AA基因型个体的肝重显著低于AB和BB基因型个体。初步推断OBR基因可能是影响鸡脂肪性状的主效基因或与主效基因连锁 ,推测可以利用这个多态位点对鸡的体脂性状进行标记辅助选择     

鸡瘦蛋白受体(OBR)基因外显子9单核苷酸多态性分析

瘦蛋白受体属于Ⅰ类细胞因子超家族受体,其在瘦蛋白的信号转导中起着关键的作用。本研究根据瘦蛋白受体基因外显子9的序列设计引物,用PCR-SSCP的方法对高脂系（fatness line,FL）、低脂系(leanness line,LL)、北京油鸡、白耳鸡、石歧杂、矮小黄鸡、小型黄鸡、惠阳胡须鸡、隐性白羽鸡和海兰蛋鸡等品种（系）进行了单核苷酸多态性分析,并检测到了多态性。对两种纯合子片段克隆和测序,结果表明在编码区的1167位碱基发生了突变,由C突变为A,但是编码的氨基酸并没有发生改变。经独立性检验,基因型频率和基因频率分布与品种有关,北京油鸡的AA基因型频率显著高于其他品种;高脂系中A基因频率显著高于低脂系。 Abstract:Leptin receptor is a type I cytokine super family member and plays an important role in leptin functioning signal transduction.This study was designed to investigate the single nucleotide polymorphism (SNP) of OBR gene in various breeds,including Fatness Line (FL),Leanness Line (LL),Beijing Youji,Baierji,Shiqiza,Dwarf Yellow Chickens,Mini Yellow Chickens,Huiyang Huxuji,Recessive White Chickens and Hyline Layer.The primers for exon 9 in OBR gene were designed from the database of chicken genomic sequence and the SNPs were detected by PCR-SSCP method.One SNP (C/A at 1167 in cds) was found among individuals within all breeds.However,the amino acid was not changed because it was a silence mutantion.The result of population genetics analyses showed that the frequency of AA genotype in Beijing Youji was significantly higher than that in other lines.Also,the frequency of A allele in FL was significantly higher than that in LL.     

利用微卫星标记分析山东地方鸡品种的遗传多样性

微卫星是近几年来应用较多的一种分子标记,可有效地进行基因鉴定与系谱分析,并可估算群体间的遗传距离。通过选用5个微卫星标记,检测了山东省5个地方鸡种：日照麻鸡、寿光鸡、莱芜黑鸡、济宁百日鸡、鲁西斗鸡以及一个外来鸡种——安卡黄鸡和一个外省地方鸡种——广西黄鸡共7个鸡种的遗传多样性。根据测试结果计算了每个等位基因的频率,以基因频率为基础分析了品种内的遗传变异和品种间的DA遗传距离,并讨论了微卫星多态性在应用于群体遗传变异及亲缘关系等方面的意义。结果表明：共检测到40个等位基因,其中等位基因数最多的位点为。ADL0136(10个);等位基因数最低的位点为ADL0146(5个);而且每个位点的等位基因分布并不均匀,都有一种或几种优势基因存在。在7个品种中,杂合度最低的为寿光鸡,杂合度值为0．3327,因为此鸡种多年来一直由寿光市慈伦种鸡场进行纯繁保种,未与其他鸡种杂交,因此杂合度最小;其他鸡种杂合度也都低于0．4,据分析可能是由于日照麻鸡、济宁百日鸡群体较小;莱芜黑鸡是正在选育的一个品种,个体间遗传关系也不远;安卡黄鸡和广西黄鸡自从引人嘉明公司后,群体近交现象普遍,因此各鸡种杂合度都偏低。由此可见,通过对杂合度的计算,微卫星可以较好地反应群体内的变异。各品种PIC值的变动范围从0．6196(寿光鸡)到0．7027(莱芜黑鸡),PIC值的大小与杂合度的高低体现了较高的一致性。对DA遗传距离的计算表明：日照麻鸡与济宁百日鸡的距离最近,而鲁西斗鸡与其他鸡种距离均较远。用UPGMA法进行聚类分析,结果7个鸡种被聚为3类：山东的4个地方鸡种寿光鸡、日照麻鸡、莱芜黑鸡与济宁百日鸡聚为一类;安卡黄鸡和广西黄鸡聚在一起;鲁西斗鸡独自为一类。这与几个鸡种的分化与选育历史是一致的,因此聚类图能够比较正确地反映7个品种之间的亲缘关系。     

利用微卫星标记分析四川8个地方鸡品种遗传多样性

通过选用30个多态性较好的微卫星标记,检测了四川省8个地方鸡品种：峨嵋黑鸡、泸宁鸡、旧院黑鸡、米易鸡、石棉草科鸡、凉山崖鹰鸡、兴文乌骨鸡、沐川乌骨鸡的遗传多样性。利用等位基因频率计算出各群体的平均遗传杂合度（H）、多态信息含量（PIC）和群体间的DA遗传距离。结果表明：30个微卫星位点中有24个微卫星位点在8个鸡群体中的多态信息含量均为高度多态,可作为有效的遗传标记用于各鸡品种的遗传多样性和系统发生关系的分析。各鸡种的杂合度都较高,平均杂合度范围是0.629~0.681,最高的是泸宁鸡（0.681）,最低的是旧院黑鸡（0.629）。据分析可能是由于交通闭塞,形成了家禽品种的多种多型,而且杂合度的高低与PIC值的大小体现了较高的一致性。对DA遗传距离的计算表明：用UPGMA法进行聚类分析,结果8个鸡品种被聚为3类：Ⅰ类：峨嵋黑鸡、米易鸡、泸宁鸡、旧院黑鸡聚为一类。米易鸡、泸宁鸡先聚在一起,然后又与峨嵋黑鸡在较近的距离聚在一起,然后再与旧院黑鸡聚在一起;Ⅱ类：石棉草科鸡、兴文乌骨鸡、沐川乌骨鸡聚为一类。兴文乌骨鸡、沐川乌骨鸡在较近的距离聚在一起,然后又与石棉草科鸡在较近的距离聚在一起;Ⅲ类：凉山崖鹰鸡独自聚为一类。这与几个鸡品种的来源、分化、选育历史及地理分布是一致的。     

Genetic Polymorphism of β-Lactoglobulin Gene in Native Turkish Sheep Breeds

The genetic polymorphism of the β-lactoglobulin gene was investigated in three native Turkish sheep breeds. The study was carried out on 108 sheep (29 Kıvırcık, 38 G?k?eada, and 41 Sakız) by means of PCR-RFLP methods. Two genetic variants (A and B) and three genotypes (AA, AB, and BB) of β-lactoglobulin have been identified. The gene frequencies of β-LG A and B were 0.7759 and 0.2241 in Kıvırcık, 0.7632 and 0.2368 in G?k?eada, and 0.9756 and 0.0244 in Sakız breeds, respectively. The populations were in Hardy–Weinberg equilibrium in all samples from the three breeds.     

56个中国地方猪种微卫星基因座的遗传多样性

采用FA0—ISAG联合推荐的27个微卫星DNA标记对56个中国地方猪品种和3个引进猪种(杜洛克猪、长白猪、大白猪)进行遗传多样性分析,通过计算等位基因频率、有效等位基因数、平均遗传杂合度、基因分化系数、多态信息含量和遗传距离并进行系统聚类分析,评估其种内遗传变异和种间遗传关系。以聚类结果为基础,将56个中国地方猪种分为12类：Ⅰ、Ⅱ和Ⅲ类都是《中国猪品种志》中的华北型猪种;Ⅳ类相当于其中的江海型猪种;Ⅴ、Ⅵ、Ⅶ、Ⅷ和Ⅸ类的品种大部分属于华中型;Ⅹ和Ⅺ类相当于华南型品种;Ⅻ类相当于西南型品种。提出保种场结合保护区是一种比较符合我国地方猪种实际状况的保种模式。研究结果可为我国地方猪种种质特性研究提供基础数据,为我国地方猪品种资源的合理保护和利用提供科学依据。     

利用微卫星DNA标记法进行5个肉用绵羊品种的多态性分析和杂种优势率预测

研究选择了与产肉性能相关的5个微卫星标记,分析了这5个位点在杜泊、德国美利奴、特克塞尔、道塞特和右玉本地绵羊5个品种中的遗传多态性.通过遗传分析预测了4个国外引进肉用绵羊品种与右玉本地绵羊的杂种优势,并与实际测定结果进行了比较分析.结果表明,利用微卫星DNA多态性进行品种间杂种优势预测是可行的,杜泊羊与右玉本地绵羊杂交的杂种优势最大,与实际测定结果一致.     

Evaluation of Bovine High-Density SNP Genotyping Array in Indigenous Dairy Cattle Breeds

In total 52 samples of Sahiwal (19 Excoffier L, Laval G, Schneider S. Arlequin 3.01: An integrated software package for population genetics data analysis. Evol Bioinform Online 2005; 1:47–50.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]), Tharparkar (17 Hayes BJ, Bowman PJ, Chamberlain, AJ, Goddard ME. Invited review: genomic selection in dairy cattle: progress and challenges. J Dairy Sci 2009; 92:433–443.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]), and Gir (16 Melka HD, Jeon EK, Kim SW, Han JB, Yoon D, Kim KS. Identification of genomic differences between Hanwoo and Holstein breeds using the Illumina Bovine SNP50 BeadChip. Genomics Inform 2011; 9:69–73.[Crossref] , [Google Scholar]) were genotyped by using BovineHD SNP chip to analyze minor allele frequency (MAF), genetic diversity, and linkage disequilibrium among these cattle. The common SNPs of BovineHD and 54K SNP Chips were also extracted and evaluated for their performance. Only 40%?50% SNPs of these arrays was found informative for genetic analysis in these cattle breeds. The overall mean of MAF for SNPs of BovineHD SNPChip was 0.248?±?0.006, 0.241?±?0.007, and 0.242?±?0.009 in Sahiwal, Tharparkar and Gir, respectively, while that for 54K SNPs was on lower side. The average Reynold’s genetic distance between breeds ranged from 0.042 to 0.055 based on BovineHD Beadchip, and from 0.052 to 0.084 based on 54K SNP Chip. The estimates of genetic diversity based on HD and 54K chips were almost same and, hence, low density chip seems to be good enough to decipher genetic diversity of these cattle breeds. The linkage disequilibrium started decaying (r²?相似文献   

我国主要地方绵羊品种mtDNA D-loop区PCR-RFLP研究

利用5种限制性内切酶（Hinf I,Msp I,Sau3A I,Xsp I,Taq I）,采用PCR-RFLP技术研究了我国9个地方绵羊品种以及2个引入品种共计83只绵羊个体线粒体DNA D-loop区的多态性。结果表明,我国主要地方绵羊品种线粒体DNA D-loop区存在两种基本单体型,提示我国主要地方绵羊品种起源于两个母系祖先。线粒体DNA D-loop区多态度为0.042 1%,说明我国地方绵羊品种线粒体DNA多态度较为贫乏。
     

Phylogenetic Relationships among Indigenous Sheep Populations in East Asia based on Five Informative Blood Protein and Nonprotein Polymorphisms

The phylogenetic relationships among 23 local sheep breeds and varieties in East Asia were determined based on allele frequency data for five informative and polymorphic blood protein and nonprotein loci (transferrin, arylesterase, hemoglobin-β, X-protein, and potassium transport) using electrophoresis and ion-densitometric techniques. Comparatively striking differences in the frequency distributions, especially of hemoglobin-β and X-protein alleles, were seen between the northern population of the Khalkhas, Bhyanglung, Baruwal, Jakar, Sakten, and five Chinese breeds and the southern population of the Bengal, Kagi, Lampuchhre, Myanmar, and Sipsu breeds. Clustering analyses using UPGMA and NJ methods and principal component analysis, using the data of all populations and loci tested, clearly demonstrated the difference. The two population groups are divided by the boundary of the Himalayas, and each diverged into three subgroups: Mongolian, Tibetan, and Himalayan groups in the north and Indian groups I, II, and III in the south. It was noted that the genetic differentiation of populations was more distinct in the northern group. These findings strongly suggest the existence of at least two large and phylogenetically different gene pools of sheep in East Asia.