猪MyoG基因的PCR-RFLP多态性分析

以杜洛克、长白、大约克、南昌白、二花脸、梅山猪、玉山黑猪、乐平花猪、金华两头乌及上高两头乌等中外10个猪种共计561头猪为研究材料,采用3对引物（PCR1、PCR2、PCR3）分别扩增猪肌细胞生成素（MyoG）基因的不同区域,扩增产物经限制性核酸内切酶MspⅠ酶切后发现：（1）在PCR1 MspⅠ-RFLP位点上,外来品种杜洛克、长白、大约克及培育品种南昌白中极大多数个体表现为AA型,个别为BB型;而6个中国地方猪种除乐平花猪外均以BB型居多。（2）在PCR2 MspⅠ-RFLP位点上,6个中国地方猪种除一头玉山黑猪表现为MN型外,其余均为MM型;而外来品种以NN型占大多数,培育品种南昌白更趋向于外来品种。（3）在PCR3 MspⅠ-RFLP位点上,所有猪种均可得到扩增产物,但无MspⅠ酶切位点。（4）在梅山猪及与其亲缘关系较近的二花脸猪中,没有发现Soumillion等(1997)报道的梅山猪特异性MspⅠ多态性酶切位点。     

Gene Flow and the Geographical Distribution of a Molecular Polymorphism in DROSOPHILA PSEUDOOBSCURA

This paper discusses the relation between the geographical distribution of an enzyme polymorphism and population structure in Drosophila pseudoobscura. California populations of this species living in very different montane and lowland habitats separated by several kilometers are similar to each other in the frequency of an esterase allele. Previous estimates suggest that gene flow is too limited to account for this homogeneity of genetic structure, so that it must reflect some balancing force of natural selection. We show, however, that dispersal over unfavorable habitats is much greater than earlier supposed. Isolated populations of D. pseudoobscura separated by 15 km from other populations are subject to large amounts of immigration. This is shown by changes in the seasonal abundance of this species and in the annual pattern of lethal alleles in such populations. The genetic structure of an experimentally perturbed isolated population in an oasis returned to normal within a single year, suggesting that such populations are ephemeral and that the oasis is subject to annual recolonization by distant migrants. Direct assessment of marked flies shows that they can move at least 10 km in 24 hours over a desert. Such extensive gene flow may help explain the distribution of the esterase allele, and is relevant to the high level of molecular polymorphism and its general lack of geographic differentiation throughout the range of D. pseudoobscura.     

小型猪胰淀素基因的多态性分析

目的探讨我国3个特有的小型猪品系,巴马小型猪,五指山小型猪,中国农大小型猪胰淀素（IAPP）基因多态性分布,为我国小型猪在2型糖尿病及代谢性疾病研究中的应用提供基础资料。方法提取3个品系小型猪血液基因组DNA,针对IAPP基因外显子3~内含子3的部分序列进行PCR扩增,产物鉴定、测序,统计分析基因多态。结果在所扩增的片段中共检测出2个SNPs位点,SNPs1：43G→A,位于外显子上,未引起的氨基酸的改变,突变发生在五指山猪（G/A杂合突变为16.7%,A纯合突变为83.3%）和中国农大猪（G/A杂合突变60%,A纯合突变为20%两个品系中。SNPs2：214C→T,位于内含子上,发生在五指山猪（T/C杂合突变为16.7%,T纯合突变为83.3%）和中国农大猪（T/C杂合突变为20%,T纯合突变为60%）两个品系中。结论在IAPP基因外显子3-内含子3的部分扩增序列中发现了2个SNPs位点,在3个品系小型猪中的分布不同。     

民猪的SM-DRB基因的PCR-RFLP多态性分析

采用克隆测序和PCR—RFLP相结合的方法,对民猪的SLA—DRB基因的整个编码区进行了扫描和多态性分析．结果表明该基因的第2外显子是整个基因的高变区,突变率达到5．6％,其中80％为有效突变,但没有发现新的等位基因;PCR-RFLP结果表明外显子1用内切酶Alu Ⅰ酶切可获得3种基因型;外显子2用内切酶TaqⅠ酶切可获得3种基因型：外显子3用内切酶BcnⅠ酶切可获得3种基因型：外显子4用内切酶MboⅠ酶切可获得2种基因型：外显子5用内切酶HinlⅠ酶切可获得3种基因型．Hardy-Weinberg平衡分析表明民猪在Alu Ⅰ和HinlⅠ处于平衡状态（P〉0．05）．在TaqⅠ、BcnⅠ和MboⅠ处于不平衡状态．     

民猪的SLA-DRB基因的PCR-RFLP多态性分析

采用克隆测序和PCR-RFLP相结合的方法,对民猪的SLA-DRB基因的整个编码区进行了扫描和多态性分析.结果表明该基因的第2外显子是整个基因的高变区,突变率达到5.6%,其中80%为有效突变,但没有发现新的等位基因;PCR-RFLP结果表明外显子1用内切酶Alu Ⅰ酶切可获得3种基因型;外显子2用内切酶Taq Ⅰ酶切可获得3种基因型;外显子3用内切酶Bcn Ⅰ酶切可获得3种基因型;外显子4用内切酶Mbo Ⅰ酶切可获得2种基因型;外显子5用内切酶Hin1 Ⅰ酶切可获得3种基因型.Hardy-Weinberg平衡分析表明民猪在Alu Ⅰ和Hin1 Ⅰ处于平衡状态(P＞0.05),在Taq Ⅰ、Bcn Ⅰ和Mbo Ⅰ处于不平衡状态.     

HLA－DRB1基因位点多态性的PCR－RFLP分析

设计并建立一套适合国内应用的改良ＰＣＲ－ＲＦＬＲ方法,分５组特异性扩增ＤＮＡ样品,随后进行酶切定型分析,准确检测了编码ＤＲ抗原特异性的ＨＬＡ－ＤＲＢ１基因位点的多态性,该法采用分组扩增,不发生与其它ＤＲＢ位点等位基因的交叉扩增,不仅适合纯合子的区分而且可以清楚准确地检测杂合子样品,已报道过的ＤＲＢ１位点编码的特异性组合都可以通过这个方法得到准确分析。所使用的Ⅱ类限制性内切酶均价格便宜、易购。     

猪精氨酸-丝氨酸蛋白激酸3(SRPK3)基因的克隆、表达及多态性分析

通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3 (serine/arginine-rich specific kinase 3,SRPK3) 的全长基因CDS区;采用生物信息学方法分析了SRPK3基因核酸序列并对其所编码的的蛋白序列进行了预测与分析编码蛋白序列的结构特点;采用PCR-SSCP方法对大白猪,野猪,民猪及野家杂交猪的SRPK3基因的多态性进行了检验;采用实时荧光定量PCR (Real-time) 方法检测了SRPK3在1日龄和30日龄大白猪及杜洛克的心脏、肌肉、脾脏、肝脏、肾脏、肺脏、胃、小肠、大肠、脑的表达情况;采用皮下注射的方式构建猪骨骼肌损伤模型用于研究在骨骼肌修复过程中SRPK3基因表达特性。经拼接所得到的1 708bp核苷酸片段,涵盖了SRPK3基因的全长CDS (1 701bp),该基因编码含567个氨基酸片段;蛋白存在两个S_TKc结构域,猪SRPK3蛋白序列与人和牛的相似性较高。PCR-SSCP检测发现第6外显子上A⁶²⁹→G⁶²⁹,T⁶⁵³→T⁶⁵³的突变,氨基酸变化为Pro→His,Ile→Thr;第9外显子处的G¹⁰⁵⁹→ A¹⁰⁵⁹,氨基酸无突变。利用荧光定量PCR研究发现,表达结果显示该基因表达具有组织和种间特异性。SRPK3基因的表达在整个骨骼肌细胞损伤修复过程中逐渐升高。SRPK3基因主要在肌肉和心肌内表达,骨骼肌损伤修复过程中伴随骨骼肌细胞分化SRPK3的表达持续升高,推测其可能与骨骼肌细胞发育相关。     

卡拉库尔羊MHC-DRB3基因的遗传多样性研究

目的:实验通过MCH-DRB3基因来探讨卡拉库尔羊的遗传多态性,为绵羊遗传资源的合理利用及保护提供理论基础和科学依据.方法:采用PCR-Clone测序方法首次对卡拉库尔羊的MHC-DRB3基因的第二外显子进行分子遗传多态性检测与分析.结果:总共检测出35种单倍型,总的单倍型多样度(Hd)为0.958,核苷酸多样性(Pi)为0.05786,平均核苷酸差异数(k)为16.49062,简约信息多态位点数为54.卡拉库尔羊MHC-DRB3基因的氨基酸序列的氨基酸组成进行分析可知含量最多的氨基酸是精氨酸(Arg),平均含量为12.9%,含量最低的氨基酸是蛋氨酸(Met),平均含量为0.018%.结论:从以上参数可以看出卡拉库尔羊具有很高的遗传多样性.     

Global Population Genetic Structure and Male-Mediated Gene Flow in the Green Turtle (Chelonia Mydas): RFLP Analyses of Anonymous Nuclear Loci

We introduce an approach for the analysis of Mendelian polymorphisms in nuclear DNA (nDNA), using restriction fragment patterns from anonymous single-copy regions amplified by the polymerase chain reaction, and apply this method to the elucidation of population structure and gene flow in the endangered green turtle, Chelonia mydas. Seven anonymous clones isolated from a total cell DNA library were sequenced to generate primers for the amplification of nDNA fragments. Nine individuals were screened for restriction site polymorphisms at these seven loci, using 40 endonucleases. Two loci were monomorphic, while the remainder exhibited a total of nine polymorphic restriction sites and three size variants (reflecting 600-base pair (bp) and 20-bp deletions and a 20-bp insertion). A total of 256 turtle specimens from 15 nesting populations worldwide were then scored for these polymorphisms. Genotypic proportions within populations were in accord with Hardy-Weinberg expectations. Strong linkage disequilibrium observed among polymorphic sites within loci enabled multisite haplotype assignments. Estimates of the standardized variance in haplotype frequency among global collections (FST = 0.17), within the Atlantic-Mediterranean (FST = 0.13), and within the Indian-Pacific (FST = 0.13), revealed a moderate degree of population substructure. Although a previous study concluded that nesting populations appear to be highly structured with respect to female (mitochondrial DNA) lineages, estimates of Nm based on nDNA data from this study indicate moderate rates of male-mediated gene flow. A positive relationship between genetic similarity and geographic proximity suggests historical connections and/or contemporary gene flow between particular rookery populations, likely via matings on overlapping feeding grounds, migration corridors or nonnatal rookeries.     

Recombination Dynamics of a Human Y-Chromosomal Palindrome: Rapid GC-Biased Gene Conversion,Multi-kilobase Conversion Tracts,and Rare Inversions

The male-specific region of the human Y chromosome (MSY) includes eight large inverted repeats (palindromes) in which arm-to-arm similarity exceeds 99.9%, due to gene conversion activity. Here, we studied one of these palindromes, P6, in order to illuminate the dynamics of the gene conversion process. We genotyped ten paralogous sequence variants (PSVs) within the arms of P6 in 378 Y chromosomes whose evolutionary relationships within the SNP-defined Y phylogeny are known. This allowed the identification of 146 historical gene conversion events involving individual PSVs, occurring at a rate of 2.9–8.4×10⁻⁴ events per generation. A consideration of the nature of nucleotide change and the ancestral state of each PSV showed that the conversion process was significantly biased towards the fixation of G or C nucleotides (GC-biased), and also towards the ancestral state. Determination of haplotypes by long-PCR allowed likely co-conversion of PSVs to be identified, and suggested that conversion tract lengths are large, with a mean of 2068 bp, and a maximum in excess of 9 kb. Despite the frequent formation of recombination intermediates implied by the rapid observed gene conversion activity, resolution via crossover is rare: only three inversions within P6 were detected in the sample. An analysis of chimpanzee and gorilla P6 orthologs showed that the ancestral state bias has existed in all three species, and comparison of human and chimpanzee sequences with the gorilla outgroup confirmed that GC bias of the conversion process has apparently been active in both the human and chimpanzee lineages.     

牦牛MC1R基因的克隆测序及其分析研究

以麦洼牦牛、斯布牦牛、天祝牦牛和九龙牦牛为研究对象,对黑色素皮质素受体1(Melanocortin receptor I,MCIR)基因编码区进行了克隆测序及分析.结果表明,牦牛的MC1R基因编码区全长954 bp,编码317个氨基酸:4个牦牛品种间及与普通牛间在MC1R基因的编码区内共有13个碱基差异,无碱基的插入和缺失现象,编码蛋白共有9个氨基酸差异.MC1R蛋白为亲水性蛋白,无信号肽,有糖基化位点和7个跨膜区.系统进化分析显示,麦洼牦牛与斯布牦牛的MC1R基因相似性最近.本研究结果时今后开展MC1R基因与牦牛毛色性状的相关性分析以及牦牛的毛色遗传机理、基因定位、基因表达调控等研究具有重要的意义.     

小型猪葡萄糖转运子4外显子4a的基因多态性分析

目的分析我国特有的3个小型猪品系巴马小型猪、中国农大小型猪、五指山小型猪葡萄糖转运子4基因外显子4a的单核苷酸多态性（SNPs）分布特点,为我国小型猪在糖尿病和代谢性疾病研究中提供基础资料。方法以3个品系小型猪基因组DNA为模板,应用特异性引物进行PCR扩增,扩增产物纯化测序,进行BLAST比对分析。结果在小型猪GLUT-4基因外显子4a上有2个SNP位点：SNP1：GCT→GCC（Ala133 Ala）,3个品系均发生了变化,均为纯合突变,其突变率为（22/22,100%）。SNP2：GGC→GGT（Gly146 Gly）,巴马小型猪突变率为（6/6,100%）,均为纯合突变;五指山小型猪突变率为（6/6,100%）,均为纯合突变;中国农大小型猪突变率为（10/10,100%）,其中包括6例纯合突变（6/10,60%）和4例杂合突变（4/10,40%）。结论SNPs1,在所有测定的小型猪品系中检出,这可能是所测小型猪的共有特征。SNPs2可能是小型猪品系之间的差异。     

A Recent Shift from Polygyny to Monogamy in Humans Is Suggested by the Analysis of Worldwide Y-Chromosome Diversity

Molecular genetic data contain information on the history of populations. Evidence of prehistoric demographic expansions has been detected in the mitochondrial diversity of most human populations and in a Y-chromosome STR analysis, but not in a previous study of 11 Y-chromosome SNPs in Europeans. In this paper, we show that mismatch distributions and tests of mutation/drift equilibrium based on up to 166 Y-chromosome SNPs, in 46 samples from all continents, also fail to support an increase of the male effective population size. Computer simulations show that the low nuclear versus mitochondrial mutation rates cannot explain these results. However, ascertainment bias, i.e., when only highly variable SNP sites are typed, may be concealing any Y SNPs evidence for a recent, but not an ancient, increase in male effective population sizes. The results of our SNP analyses can be reconciled with the expansion of male effective population sizes inferred from STR loci, and with mitochondrial evidence, by admitting that humans were essentially polygynous during much of their history. As a consequence, until recently only a few men may have contributed a large fraction of the Y-chromosome pool at every generation. The number of breeding males may have increased, and the variance of their reproductive success may have decreased, through a recent shift from polygyny to monogamy, which is supported by ethnological data and possibly accompanied the shift from mobile to sedentary communities.     

Complex Evolution of a Y-Chromosomal Double Homeobox 4 (DUX4)-Related Gene Family in Hominoids

The human Y chromosome carries four human Y-chromosomal euchromatin/heterochromatin transition regions, all of which are characterized by the presence of interchromosomal segmental duplications. The Yq11.1/Yq11.21 transition region harbours a peculiar segment composed of an imperfectly organized tandem-repeat structure encoding four members of the double homeobox (DUX) gene family. By comparative fluorescence in situ hybridization (FISH) analysis we have documented the primary appearance of Y-chromosomal DUX genes (DUXY) on the gibbon Y chromosome. The major amplification and dispersal of DUXY paralogs occurred after the gibbon and hominid lineages had diverged. Orthologous DUXY loci of human and chimpanzee show a highly similar structural organization. Sequence alignment survey, phylogenetic reconstruction and recombination detection analyses of human and chimpanzee DUXY genes revealed the existence of all copies in a common ancestor. Comparative analysis of the circumjacent beta-satellites indicated that DUXY genes and beta-satellites evolved in concert. However, evolutionary forces acting on DUXY genes may have induced amino acid sequence differences in the orthologous chimpanzee and human DUXY open reading frames (ORFs). The acquisition of complete ORFs in human copies might relate to evolutionary advantageous functions indicating neo-functionalization. We propose an evolutionary scenario in which an ancestral tandem array DUX gene cassette transposed to the hominoid Y chromosome followed by lineage-specific chromosomal rearrangements paved the way for a species-specific evolution of the Y-chromosomal members of a large highly diverged homeobox gene family.     

中国荷斯坦牛线粒体DNA全序列多态性分析和分型

目的：阐明中国荷斯坦牛线粒体DNA全序列多态性及其起源关系。方法：从上海松江科研基地的10头中国荷斯坦牛外周血中抽提DNA,设计引物扩增全长线粒体DNA,通过对其进行序列测定、分析,绘制系统进化发育树。结果：得到了中国荷斯坦牛线粒体基因组的全序列和个体比对结果等相关统计数据,对多态性位点和突变位点等遗传信息进行了序列分析,并进一步分型。结论：研究结果对提高中国荷斯坦牛体细胞核移植和线粒体DNA多态性高通量分析效率有一定的指导意义,为线粒体DNA多态性检测芯片的研究奠定了基础。     

10个牛品种线粒体12S rRNA基因多态性分析

以鲁西牛、渤海黑牛、大别山牛、南阳牛、晋南牛、秦川牛、中国西门塔尔牛、日本和牛、海福特牛和安格斯牛10个牛品种共计353头牛为研究对象,利用单链构象多态性（SSCP）分子标记方法对其线粒体DNA 12S rRNA基因进行了多态性分析,结果发现该基因出现分布频率不同的3种单倍型（Ⅰ、Ⅱ、Ⅲ）,其中单倍型Ⅰ出现的频率最低,仅在晋南牛1个个体中发现;单倍型Ⅱ在中国本地黄牛中都存在,在中国西门塔尔牛中的分布频率很低,但在3个国外品种海福特牛、安格斯牛和日本和牛中不存在;单倍型Ⅲ在这10个牛品种中均有分布,但以国外品种最丰富。计算平均多态信息含量,发现我国地方品种在0.232～0.423之间,基本属于中度多态,说明我国地方黄牛品种在12S rRNA上多态性比较丰富,国外品种较为贫乏;但是单倍型Ⅰ频率很低有丢失的趋势,需要对其进行保护。     

RAPD Analysis of DNA Polymorphism in Turkish Sheep Breeds

The genetic diversity and phylogenetic relationships of 108 individual sheep from three Turkish sheep breeds (K?v?rc?k, Gökçeada, and Sak?z) were studied using RAPD analysis. Polymorphisms within and between populations were assayed using 15 random primers, and 82 loci were amplified ranging from 250 to 2,500 bp. The percentage of polymorphic loci was found to be 80.49, 78.05, and 73.17%, for K?v?rc?k, Gökçeada, and Sak?z sheep breeds, respectively. Total genetic diversity was 0.2265, and the average coefficient of genetic differentiation was 0.1181. Genetically, the Gökçeada breed was more closely related to the Sak?z breed than to the K?v?rc?k breed.     

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.