从江香猪DRD1基因多态及其生物信息学分析

为揭示猪DRD1基因多态性,本研究以从江香猪为研究对象,以外三元(杜×长×大)杂交猪及贵州宗地花猪为对照,采用DNA池和直接测序技术,筛查DRD1基因所有外显子区SNPs位点;利用生物信息学软件估算等位基因频率并预测SNPs位点对m RNA二级结构的影响。结果表明,在DRD1基因中共筛查到1个SNPs位点,位于5'UTR区为T-182G;从等位基因频率角度分析,从江香猪与贵州宗地花猪估算得到的等位基因频率较一致,与外三元(杜×长×大)杂交猪却存在较大差异;利用RNA secondary structure prediction软件预测,发现m RNA二级结构发生明显变化     

贵州三个地方猪种的系统发育分析

采用PCR产物直接测序方法首次测定贵州3个地方猪种(白洗猪、从江香猪、黔北黑猪)共计57个个体线粒体DNA控制区的全序列,将它们与GenBank上已知的25个国内外猪种一起计算遗传距离,并构建系统发育树。结果表明,3个贵州地方猪种mtDNA D-loop区的序列长度为1 254-1 314 bp之间,贵州地方猪种遗传距离在0.002 9-0.007 2之间,白洗猪与黔北黑猪、从江香猪之间的距离最大,均为0.007 2。系统发育分析的结果支持中国家猪分别有南北两个母系起源的观点,且各地方猪种之间可能有基因交流。     

利用微卫星标记分析七品种（类群）小型猪的遗传多样性

采用世界粮农组织（FAO）和国际动物遗传学会（ISAG）推荐的27个微卫星,对久仰香猪、剑白香猪、从江香猪、环江香猪、黑香猪（贵州省种猪场）、五指山猪和滇南小耳猪等我国7小型猪种（类群）及杜洛克、长白和大白等3个外来猪种的群内遗传变异性和群间遗传差异进行了分析。结果表明,7个小型猪品种（类群）均有较高的群内遗传变异,但久仰、剑白、从江和环江4个香猪类群的群内遗传变异（平均多态信息含量（PIC）为0.61～0.64）显著低于其他3个小型猪种（平均PIC为0.80～0.84）。久仰、剑白、从江和环江4个香猪类群彼此间的遗传差异较小（奈氏标准遗传距离为0.12~0.22）,但它们与其他3个小型猪种有较大的遗传差异（奈氏标准遗传距离为1.61～1.96）,与3个外来猪种的遗传差异更大（奈氏标准遗传距离为1.99～3.30）。通过聚类分析,可将这些猪种清晰地分为3大类,久仰、剑白、从江和环江4个香猪类群紧密地聚为一类,其他3个小型猪种聚为一类,3个外来猪种聚为另一类。这一结果基本符合这些猪种的地理分布和品种来源。
     

贵州从江侗族威宁彝族、荔波瑶族谷胱甘肽S-转移酶M l、Tl 和Pl 的基因多态性研究

目的：研究贵州从江侗族、威宁彝族、荔波瑶族的GSTs基因多态性。方法：在隔离自然人群中,采用多重等住基因特异聚合酶链反应方法分析GSTM1和GSTT1基因多态性,同时采用PCR-RFLP的方法和TaqMan-MGB探针基因分型方法分析GSTP1（A1578G）基因多态性。结果：贵州从江侗族、成宁彝族、荔波瑶族的GSTM1和GSTT1纯合缺失基因型频率分别为59．6％～71．2％、39．4％～72.5％。其GSTP1（A1578G）基因型频率分别为：野生型（AA）为63.3％～75％、杂合子（AG）为23.2％～35．8％、纯合突变型（GG）为0～1.9％。等位基因频率：A为81．2％～86．6％,G为13．4％～18.8％。结论：贵州从江侗族、威宁彝族、荔波瑶族的GSTM1纯合缺失基因型频率在民族间差异无统计学意义,GSTP1（A1578G）基因型频率和等住基因频率在民族间差异无统计学意义,且其等位基因频率均符合Hardy-Weinberg平衡,但其GSTT1纯合缺失基因型频率在民族间差异有统计学意义（P〈0．05）。     

宗地花猪和从江香猪ADRP基因多态性及生物信息学分析

为研究宗地花猪和从江香猪ADRP基因的单核苷酸多态性,为选种选育提供理论参考,试验以宗地花猪和从江香猪为对象,构建DNA池,采用PCR产物测序法对ADRP基因8个外显子进行单核苷酸多态性检测,并利用生物信息学方法对ADRP基因编码产物进行结构功能预测。结果显示,在两个猪种ADRP基因中筛查到5个SNPs,分别是T37C、T140C、G777A、A1061G、A1117G,其中T37C位于非编码区内,T140C和A1061G为同义突变,G777A(Val→Ile)和A1117G(Asn→Ser)为错义突变;突变前后ADRP基因m RNA二级结构、编码蛋白二级结构及三级结构均发生了变化。研究结果表明,宗地花猪和从江香猪ADRP基因具有较高的遗传多样性,可为猪种的选育和创新提供参考。     

贵州地方猪种及三元商品猪氟烷基因的检测

旨在对贵州地方猪种"抗应激敏感基因专门化品系"的培育提供技术支撑,运用PCR-RFLP技术对白洗猪、黔北黑猪、宗地花猪、糯谷猪和从江香猪5个地方猪种及三元商品猪共151份样本进行了氟烷基因检测,筛选出猪群中应激敏感基因隐性携带型HalNn、抗应激敏感基因型HalNN和应激敏感基因型Halnn。结果表明,贵州5个地方猪种全为HalNN型;三元商品猪群无Halnn型,HalNn型检出率为52.63%,Haln型等位基因频率为26.32%。     

4个贵州地方猪品种MKLN1基因中SNP位点的多态性研究

本研究在香猪和柯乐猪单核苷酸多态性(single nucleotide polymorphism,SNP)芯片筛查的基础上,采用AS-PCR技术,以香猪、柯乐猪、糯谷猪、黔北黑猪4个贵州地方猪品种为研究对象,研究muskelin 1基因第18外显子中rs81213998(DIAS0003365)位点的多态性与体尺指标之间的关系。结果显示,MKLN1第18外显子中rs81213998位点基因型在4个猪群中有多态性。香猪的C等位基因频率明显高于其它3个猪品种的相应值(p0.01),T等位基因的频率较低(p0.01),黔北黑猪、糯谷猪和柯乐猪的C和T之间的差异不明显(p0.05)。相关性分析显示,rs81213998位点与猪的管围、体长、体高、体宽呈弱的正相关。推测MKLN1基因的rs81213998位点可能与贵州地方猪品种的体尺指标之间有一定的关系。     

贵州荔波封闭人群MTHFR基因多态性研究分析

目的 对贵州汉族、布依族亚甲基四氢叶酸还原酶（Methylenetetrahydrofolate Reductase,MTHFR）基因多态性进行研究,为贵州少数民族基因多态性数据库的建立提供相关数据。方法 应用聚合酶链式反应及限制性片段长度多态性检测贵州荔波汉族90例、布依族119例MTHFR基因两个单核苷酸（677及1298位）多态位点的基因频率及基因型频率。结果 汉族、布依族MTHFR 677位T等位基因的分布频率分别是22、8％,16．1％,x^2=1．561,P〉0．1;MTHFR 1298位C等位基因的分布频率分别是28．9％,39、1％,x^2=2．075,P〉0．1;677CT／1298AC双杂合子的分布频率分别是16．66％,22．7％。结论 MTHFRC 677T和A1298C多态性在中国南方和北方人群存在群体差异;贵州汉族与布依族此两位点无显著性差异。贵州荔波布依族MTHFR 1298位有较高的C等位基因频率。     

贵州从江香猪FoxO4、PRKAA1基因在不同组织中的表达分析

为研究FoxO4、PRKAA1基因在从江香猪不同组织中的表达情况,本实验采用实时荧光定量PCR技术,检测FoxO4、PRKAA1基因的mRNA表达量,分析FoxO4、PRKAA1在贵州从江香猪不同组织的差异表达。结果显示,FoxO4基因mRNA在从江香猪所检测的9个组织样中均有表达,其中在脂肪组织中表达量最高,且差异极显著(p0.01),在背最长肌组织中表达量最低;PRKAA1基因mRNA在从江香猪所检测的9个组织样中也均有表达,其中在肺组织中表达量最高,且差异极显著(p0.01),在脂肪组织中表达量次之,在背最长肌组织中表达量最低。FoxO4、PRKAA1基因可能在肌内脂肪(IMF)沉积中发挥重要作用,为进一步研究FoxO4、PRKAA1的功能奠定基础。     

迪庆藏猪的遗传多样性研究

采用水平板淀粉凝胶电泳法检测了52头迪庆藏猪的13个血液蛋白座位的多态性,并计算各座位等位基因的频率及其估计误差以及基因频率估计值的精确度和可靠性。结果发现,迪庆藏猪在Tf、Cp、Am、Hp、6PGD、CEs、Ca共7个座位表现出多态性,并分别受控于3、2、5、5、2、2、3个常染色体等位基因。其余6个座位(Pa 、EsD、 PHI、 PGM、 G6PD、 MDH)未检测出多态性。     

Comparative porcine gene mapping relative to human chromosomes 9, 10, 20 and 22

Comparative anchor tagged sequence (CATS) consensus primers from loci mapped to human chromosomes 9, 10, 20, and 22 have been used to amplify homologous loci in pigs. Of 53, CATS primers tested in pigs, only 23 yielded products homologous to the human locus (42% success). Ten loci were physically mapped (43% success rate for verified products, but only 19% for primers tested). Due to lack of polymorphism, linkage mapping was possible only for AMBP. Map locations were consistent with human/pig ZOO-FISH, except for ADRA1A, whose position is still equivocal in humans. These CATS primers have made very limited contributions to pig/human comparative gene mapping because of low efficiency of amplification of orthologous porcine product, frequent amplification from rodent template in a somatic hybrid panel and low level of polymorphism.     

Cloning and characterization of highly polymorphic porcine microsatellites.

A size-fractionated (200-400 bp) porcine genomic library was screened with the dinucleotide motifs (TG)n and (TC)n. The number of TG- and TC-positive clones was 83 and four, respectively, implying that the former motif is more frequent in the porcine genome, as previously reported in other species. Twenty-six TG-clones were sequenced, and the number of repeats varied between 16 and 42 with different compositions of the repetitive sequences; 17 clones had a perfect stretch of TG-repeats, four had imperfect stretches, and five had a compound structure with TG-repeats followed by TC-repeats. Primers for DNA amplification using the polymerase chain reaction (PCR) were synthesized for six loci. Ten unrelated individuals (two wild boars and eight domestic pigs of the Swedish Yorkshire breed) were screened for microsatellite polymorphism. All six microsatellite loci were polymorphic with two to seven alleles and observed heterozygosities in the range of 0.42-0.84; the inheritance of the observed polymorphism was confirmed by family studies. The characteristics of microsatellites make them highly suitable as genetic markers, and these microsatellites were isolated as a part of a pig gene mapping project.     

北京黑猪FSHb 亚基基因的多态性与繁殖性状的关联分析

本研究以北京黑猪为研究对象, 以FSHb 亚基基因为产仔性状的候选基因, 分别采用PCR产物直接电泳和PCR-RFLP方法来检测FSHβ亚基基因2个位点的多态性。结合测序发现: FSHb-1位点上, 北京黑猪BB型的134与135 bp (D00621序列的6 473与6 474 bp) 之间插入273 bp的片段而产生多态, 序列分析表明该插入片段为一典型的逆转座子, 在插入片段中还发现了一个RNA 聚合酶Ⅲ内部启动子; FSHb-2位点上, 由于扩增片段173 bp处存在C→T的突变, 使得HaeⅢ酶切位点消失而产生多态; 2个位点的A、B等位基因在北京黑猪群体中都有分布, 且处于低度多态。χ2适合性检验结果表明, 该群体在这2个位点的突变都达到Hardy-Weinberg平衡状态 (P>0.05)。用SAS 8.2 软件最小二乘法拟合线性模型, 将基因座不同基因型与繁殖性状总产仔数 (TNB)、产活仔数 (NBA) 和出生重 (WB) 进行了关联分析, 结果表明: 就初产母猪而言, FSHb-1位点上, AA型比AB和BB型个体的TNB分别多0.96头和1.85头 (P＜0.05), AA和AB型比BB型个体的NBA分别多0.95头和1.69头(P＜0.05)。FSHb-2位点上, AA型比AB型和BB型个体的TNB分别多1.57头和2.15头 (P＜0.05); AA和AB型比BB型个体的NBA分别多1.00头和0.94头 (P＜0.05); 就经产母猪而言, FSHb-2位点上, AA型个体的WB比BB型的WB重0.25 kg (P＜0.05)。全部群体的FSHb-1位点的A等位基因和初产母猪FSHb-2位点的A等位基因对TNB、NBA和WB表现为正效应。     

BsaH Ⅰ对猪激素敏感脂肪酶基因外显子Ⅰ的PCR-RFLP分析

以华北野猪、东北野猪和山西黑猪、长白猪、大白猪、马身猪共计287头猪作为研究对象,对其HSL基因外显子Ⅰ区域进行了PCR-RFLP多态性研究,发现不同品种猪间存在多态性。瘦肉型大白猪、长白猪全部表现为GG基因型;山西黑猪表现为AA、AG和GG三种基因型;脂肪型地方猪种马身猪为单一的AA基因型;华北杂种野猪、东北纯种野猪及杂种野猪表现为AG和GG两种基因型。等位基因A、G及三种基因型的频率在不同猪种中不同。该研究首次对华北及东北野猪HSL基因进行了多态性研究,丰富了国内外对野猪的分子生物学研究,为野猪遗传资源的合理开发利用提供了依据。     

Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

Domestication does not narrow MHC diversity in Sus scrofa

The Major Histocompatibility Complex (MHC) is a multigene family of outstanding polymorphism. MHC molecules bind antigenic peptides in the peptide-binding region (PBR) that consists of five binding pockets (P). In this study, we compared the genetic diversity of domestic pigs to that of the modern representatives of their wild ancestors, the wild boar, in two MHC loci, the oligomorphic DQA and the polymorphic DRB1. MHC nucleotide polymorphism was compared with the actual functional polymorphism in the PBR and the binding pockets P1, P4, P6, P7, and P9. The analysis of approximately 200 wild boars collected throughout Europe and 120 domestic pigs from four breeds (three pureblood, Pietrain, Leicoma, and Landrace, and one mixed Danbred) revealed that wild boars and domestic pigs share the same levels of nucleotide and amino acid polymorphism, allelic richness, and heterozygosity. Domestication did not appear to act as a bottleneck that would narrow MHC diversity. Although the pattern of polymorphism was uniform between the two loci, the magnitude of polymorphism was different. For both loci, most of the polymorphism was located in the PBR region and the presence of positive selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous substitutions in the PBR. P4 and P6 were the most polymorphic binding pockets. Functional polymorphism, i.e., the number and the distribution of pocket variants within and among populations, was significantly narrower than genetic polymorphism, indicative of a hierarchical action of selection pressures on MHC loci.     

西藏小型猪线粒体D-loop区及微卫星多态性的遗传学分析

目的通过分析西藏小型猪线粒体控制区(D-loop区)及微卫星位点的遗传多态性,检测西藏小型猪的遗传背景,从而为其作为实验动物提供分子生物学方面的可靠依据。方法利用特异性引物对西藏小型猪的线粒体D-loop区及10个具有多态性的微卫星位点进行扩增,割胶纯化并对线粒体D-loop区进行测序,另外采用聚丙烯酰胺凝胶电泳的方法分离微卫星位点的等位基因。结果西藏小型猪线粒体D-loop区全序列没有多态性,微卫星位点则具有高度的遗传多态性和杂合度,分别为0.584和0.573。结论西藏小型猪线粒体基因组无多态性,证明其在母系进化和遗传上与其他猪种较为一致,本实验所用的西藏小型猪生长于一个封闭的环境,导致其微卫星位点遗传多态性的中低度水平。     

Evaluation of the genetic diversity of Laiwu pigs using twenty-seven microsatellite markers

The Laiwu pig, an indigenous pig breed known for extremely high intramuscular fat content, is a well-preserved ancient breed due to long-term natural and artificial selections. In this study, using 27 microsatellite markers jointly recommended by the International Society of Animal Genetics (ISAG) and the Food and Agriculture Organization (FAO), we investigated the genetic diversity of the Laiwu pig breed. The genetic diversity of Laiwu pigs is dramatically low, with the observed heterozygosity ranging from 0.067 to 0.767. Among the 27 microsatellite markers, 10 were high polymorphic loci, 10 were moderate polymorphic loci, six were low polymorphic loci, and no polymorphism was detected at one locus (IGFI). Further analyses with the 10 high polymorphic loci and five moderate polymorphic loci revealed that the Laiwu pig breed was inbred and heterozygous deficient to some extent, but not severely, and that the Laiwu pigs were relatively pure, with almost no hybridization with other breeds. Two subgroups of the current 13 Laiwu pig pedigrees were identified. These results suggest that the Laiwu pig breed has a low diversity and a conservation program must be developed to preserve the “Laiwu pig” gene pool.     

Genetic Diversity of Chinese Native Pigs Inferred from Protein Electrophoresis

We examined protein polymorphism of 20 nativepig breeds in China and 3 introduced pig breeds. Thirtyloci have been investigated, among which six loci werefound to be polymorphic. Especially, the polymorphismof malate dehydrogenase (MDH), adenylate kinase(AK), and two new alleles of adenosine deaminase (ADA)had not been reported in domestic pigs and wild pigs.The percentage of polymorphic loci (P), the meanheterozygosity (H), and the mean number ofalleles (A) are 0.200, 0.065, and 1.300, respectively.The degree of genetic variability of Chinese pigs as awhole was higher than that of goats, lower than thatof cattle and horses, and similar to that ofsheep. Using the gene frequencies of the 30 loci, Nei'sgenetic distance among the 20 native breeds in China and3 introduced pig breeds was calculated by theformula of Nei. The program NEIGHBOR in PHYLIP3.5c was chosen to construct an UPGMA tree and a NJtree. Our results show that, of the total geneticvariation found in the native pig breeds in China, 31%(0.31) is ascribable to genetic differencesamong breeds. About 69% of the total genetic variationis found within breeds. Most breeds are in linkagedisequilibrium. The patterns of genetic similaritiesbetween the Chinese native pig breeds were notin agreement with the proposed pig type classification.     

苏姜猪OLR1基因多态性及其与肉质性状的关联分析

探讨OLR1基因在苏姜猪群内的遗传多态性,以及该基因多态对苏姜猪猪肉质性状的影响。采用PCR-RFLP技术检测OLR1基因在苏姜猪试验群体中的PstⅠ酶切遗传多态性,运用单因素方差分析方法分析了该多态位点对苏姜猪肉质性状的影响。结果发现,苏姜猪试验群体OLR1基因内含子5区域内发现一个PstⅠ酶切多态性,检测到CC、CD和DD三种基因型,多态信息含量呈现中度多态性。CC型与DD型个体的肌肉失水率、大理石纹间的差异达到显著水平(P<0.05),CD型个体用色差仪测得的b值显著高于DD型(P<0.05)。因此,检测到的OLR1基因PCR-RFLP-PstⅠ多态性与大理石纹等肉质性状存在着显著的相关关系,可以作为肉质性状候选基因在苏姜猪的持续选育中加以应用。