中国荷斯坦牛和鲁西黄牛mtDNAD-loop序列多态性分析

为了从母系遗传角度深入阐明中国荷斯坦牛和鲁西黄牛的群体遗传多样性以及起源进化,本研究采用PCR测序法测定了9头中国荷斯坦牛和11头鲁西黄牛的线粒体DNA D-loop区的部分序列,并经剪切后进行生物信息学软件分析.结果表明,20个个体D-loop区共711 bp,共检测到19种单倍型和50个多态位点.核苷酸多样性(Pi)为0.021 33±0.004 54,单倍型多样性(Hd)为0.994±0.019,平均核苷酸差异数(k)为15.146 20.构建的NJ网络进化树共分为两大支系,其中部分鲁西黄牛与瘤牛聚为一支,而中国荷斯坦牛和部分鲁西黄牛与普通黄牛聚为一支.说明中国荷斯坦牛和鲁西黄牛群体遗传多样性均较高;鲁西黄牛同时含有瘤牛和普通黄牛的血统,而中国荷斯坦牛只含有普通黄牛的血统.     

秦川牛和中国荷斯坦牛POU1F1基因多态性研究

采用PCR-RFLP技术研究了秦川牛(QQ)和中国荷斯坦牛(HC)共计218头个体POU1F1基因的多态性。结果表明: 秦川牛及中国荷斯坦牛群体POU1F1-HinfⅠ基因座的451 bp 的PCR产物经限制性酶HinfⅠ消化后表现多态, 其等位基因A/B频率分别为0.232/0.768、0.132/0.868; 两个群体AA、AB和BB 3种基因型的频率分别为0.030/0.403/0.567、0.007/0.251/0.742。在该基因座秦川牛群体处于Hardy-Weinberg平衡状态, 中国荷斯坦牛群体处于不平衡状态。它们在该基因座的杂合度、有效等位基因数、Shannon信息熵、多态信息含量分别为0.356/1.553/0.541/0.292、0.229/1.297/0.390/0.203; 秦川牛群体的位点杂合度、有效等位基因数、Shannon信息熵、多态信息含量均大于中国荷斯坦牛群体。     

牛黑素皮质素受体1(MCIR)基因与毛色表型的研究

牛MC1R基因不仅与毛色有关,而且与牛乳中乳蛋白的含量有关。利用PCR-RFLP和DNA测序技术分析了中国荷斯坦黑白花牛,中国荷斯坦红白花牛,鲁西黄牛和渤海黑牛共4个品种的MC1R基因。共检测出3种等位基因(ED,E ,e)。中国荷斯坦黑白花牛主要是ED和E 等位基因(ED=0.12、E =0.80);渤海黑牛也主要是ED和E 等位基因(ED=0.52、E =0.47);中国荷斯坦红白花牛和鲁西黄牛大多为e等位基因(e=0.95)。中国荷斯坦红白花牛和鲁西黄牛还存在E /e基因型。由此推测ED和E 等位基因导致黑色素合成。另外发现牛MC1R基因编码区725处存在一重要的SNP(单核苷酸多态性)。     

我国体细胞克隆牛的研究进展

近年来,由于体细胞克隆技术在畜牧业生产、疾病治疗、生物学基础理论研究及濒危动物保护等诸多领域所蕴藏的巨大应用价值,克隆效率成为科学界研究的热点问题。本文对克隆技术的相关影响因素进行了综述,研究表明,不同细胞系影响到体细胞克隆牛效率,胎儿细胞和颗粒细胞重构胚的囊胚发育率、犊牛成活率比成年成纤维细胞高。关于卵母细胞和克隆胚胎冷冻保存的研究表明,玻璃化冷冻法可以用于克隆胚胎以及卵母细胞的冷冻保存。在此基础上,讨论了国际上哺乳动物体细胞克隆研究新进展和体细胞克隆的效率,指出体细胞克隆中亟待解决的问题。     

荷斯坦牛HSP70-1基因遗传多态性与乳腺炎抗性关系

以253头中国荷斯坦奶牛为研究对象, 检测HSP70-1基因的多态性, 并分析其多态性与中国荷斯坦牛体细胞评分(Somatic cell score, SCS)的相关性。首先以PCR-SSCP法寻找HSP70-1基因编码区的突变, 并通过测序确定突变的类型, 根据突变类型寻找合适的内切酶, 最终采用PCR-RFLP方法鉴定实验牛基因型; 然后分析基因多态性与中国荷斯坦牛SCS的相关性。结果表明HSP70-1基因的1 623 bp处产生G→A→C突变, 2 409 bp处产生G→A突变, 两位点都是沉默突变, 未引起氨基酸序列的改变; 经χ2 适合性检验, 中国荷斯坦牛在两个位点均未达到Hardy-Weinberg平衡状态; 同时, 群体基因座不同基因型与SCS相关分析的结果表明, 2409位点基因型与SCS相关性不显著(P>0.05), 1623位点基因型与SCS相关性显著(P<0.05), CC型SCS显著低于AG、GG型(P<0.05), CC基因型为乳腺炎抗性基因型。在中国荷斯坦奶牛群体中, HSP70-1基因CC基因型可作为改良奶牛乳腺炎抗性性状的分子遗传标记。     

牛α辅肌动蛋白(α-Actinin)1 基因遗传变异与产犊数性状的相关性

为了探讨辅肌动蛋白1(α-actinin1)基因对母牛产犊数的影响,以鲁西单胎牛,鲁西双胎牛,南阳牛,晋南牛,荷斯坦牛,三河牛和延边牛为研究对象,以α-actinin1为影响产犊数的候选基因,分别扩增418 bp和505 bp 2个片段,采用直接测序,RFLP-RsaⅠ和RFLP-Apa Ⅰ方法检测α-actinin1基因的多态性,并将其与产犊数性状进行了关联分析.在内含子15 第227 nt处的碱基发生G→A突变,和内含子10第3 124 nt处发生A→G突变,使其产生酶切多态.对2个酶切多态位点进行基因型分型,χ2检验表明：在G227A位点,除了鲁西单胎牛外,其他群体都已达到Hardy-Weinberg平衡;在A3 124G位点,除了鲁西双胎牛外,其余群体都已经达到Hardy-Weinberg平衡.SAS 9.0的最小二乘拟和一般线性模型分析结果表明：A3 124G位点的AG基因型的产犊数的最小二乘均数极显著（P＜0.01）高于基因型AA;而单倍型组合G G的产犊数的最小二乘均数显著高（P＜0.01）于其它3种单倍型组合.α-actinin1基因有可能作为产犊数性状的候选分子的遗传标记.     

牛RXRG基因遗传变异与双胎性状的关联分析

以视黄素X受体基因g(retinoid X receptor-gamma, RXRG)作为牛双胎性状的候选基因, 运用测序法寻找牛RXRG基因SNPs, 筛查到一个新的多态位点A1941G, 该位点位于3′UTR。运用PCR-RFLP法验证并分析该位点在鲁西牛双胎群体和单胎群体及中国西门塔尔牛、安格斯牛和西蒙杂交牛单胎群体间的多态性, 结果表明, 在鲁西牛双胎和单胎群体中分布A、B两个等位基因,处于中度多态。经χ2适合性检验, 鲁西双胎牛群体在该位点未达到Hardy-Weinberg平衡状态(P < 0.05)。将鲁西牛群体的A1941G位点的基因型效应与双胎性状进行关联分析, 卡方独立性检测结果显示, 基因型分布在鲁西单、双胎牛群体上差异达到极显著水平(P < 0.01)。     

牛黑素皮质素受体1(MC1R)基因与毛色表形的研究

牛MC1R基因不仅与毛色有关, 而且与牛乳中乳蛋白的含量有关。利用PCR-RFLP和DNA测序技术分析了中国荷斯坦黑白花牛, 中国荷斯坦红白花牛, 鲁西黄牛和渤海黑牛共4个品种的MC1R基因。共检测出3种等位基因(E^D, E⁺, e)。中国荷斯坦黑白花牛主要是E^D和E⁺等位基因(E^D=0.12、E⁴=0.80); 渤海黑牛也主要是E^D和E⁺等位基因(E^D=0.52、E⁺=0.47); 中国荷斯坦红白花牛和鲁西黄牛大多为e等位基因(e=0.95)。中国荷斯坦红白花牛和鲁西黄牛还存在E⁺/e基因型。由此推测E^D和E⁺等位基因导致黑色素合成。另外发现牛MC1R基因编码区725处存在一重要的SNP(单核苷酸多态性)。     

CXCR2基因多态性与奶牛乳房炎和乳品质的关联

采用PCR-SSCP技术研究了荷斯坦牛、西门塔尔牛和通江黄牛3个品种共160头个体CXCR2基因多态性与乳房炎抗性性状和对牛奶品质性状的遗传效应。结果表明: CXCR2基因有3个SNP多态位点, 分别位于序列的第685 bp、777 bp和861 bp位点, 确定了5个等位基因A、B、C、E和F。685 bp位点表现为BC和CC基因型, 777 bp位点表现为AA和AB基因型, 861 bp位点为CC和BC基因型。与奶牛乳房炎敏感性有关的基因型主要是BC、CC和FF, 可能对乳房炎有抗性的是AA、AB和EE基因型。据不同基因型对乳品质的遗传效应分析来看, AA、AB和EE基因型的乳品质性状极显著或显著优于其他基因型。     

TNF-α基因多态性及其与奶牛乳房炎的相关性分析

以417头中国荷斯坦奶牛为研究对象,根据体细胞评分(Somatic cell score,SCS)的大小将该奶牛群体划分为感染牛群(100头)和健康牛群(317头)。通过PCR-RFLP和CRS-RFLP方法检测了肿瘤坏死因子α(Tumor necrosis factor-alpha,TNF-α)基因在荷斯坦奶牛群体中的多态性,并分析这些多态位点和奶牛乳房炎的相关性。研究发现了3个单核苷酸多态位点(Single-nucleotide polymorphism,SNP):第2外显子39bp处G→A的突变;第4外显子293bp处C→T的突变;5′侧翼区(5′-flanking region,5′UTR)C→G的突变。这3个突变位点分别是DraⅠ、AfaⅠ和DdeⅠ限制性内切酶的酶切多态位点,其中DraⅠ为创造酶切位点。经过基因型分析与χ2检验表明:3个酶切多态位点在荷斯坦奶牛群中均未达到Hardy-Weinberg平衡状态。运用SPSS13.0软件,采用最小二乘拟合线性模型分析3个酶切多态位点与SCS的关系,结果表明:AA基因型个体在DraⅠ酶切位点中的SCS显著大于BB及AB基因型个体(P0.05),BB基因型表现出乳房炎抗性。AfaⅠ酶切位点中BB基因型个体的SCS显著大于AA及AB基因型个体(P0.05),AA基因型表现出乳房炎抗性。DdeⅠ酶切位点中,AB基因型个体的SCS显著低于AA基因型个体(P0.05),AB基因型为优良基因型。因此BB、AA、AB基因型分别为DraⅠ、AfaⅠ、DdeⅠ酶切位点中的优良基因型,可作为分子标记应用于奶牛乳房炎抗性筛选。     

Analysis of somatic mutations at human minisatellite loci in tumors and cell lines

Hypervariable human minisatellite loci show a substantial level of germline instability, and spontaneous mutation rates to new length alleles have been measured directly by pedigree analysis. We now show that mutation events altering the number of minisatellite repeat units are not restricted to the germline, but also arise in other tissues. Mutant alleles can be detected at a very low frequency in lymphoblastoid cell lines and at much higher frequencies in clonal tumor cell populations, most particularly in gastrointestinal adenocarcinomas. Mutant alleles in these tumors are usually present at a dosage equal to or greater than that of the progenitor allele, indicating that most or all of the tumor cells carry the same clonally derived mutant allele. As with germline mutation, the incidence of somatic mutations in tumors varies from locus to locus, with the same locus showing the highest level of germline and somatic instability. Most length changes, as those in the germline, are of only a few repeat units; however, very large changes are also observed, implying that such mutations can occur in the absence of meiosis.     

7个微卫星座位与北京荷斯坦母牛体细胞评分关系的研究

选择与体细胞评分紧密连锁的7个微卫星座位BM1818、BM1258、BM1443、BM1905、BM302、BM4505和CYP21,用非变性聚丙烯酰胺凝胶电泳分析其在240头北京荷斯坦母牛中的遗传变异。计算了7个微卫星座位的等位基因频率、杂合度、多态信息含量和有效等位基因数,并利用最小二乘法拟合线性模型初步探索了这7个微卫星座位与北京荷斯坦母牛体细胞评分的关系。结果表明：微卫星BM1818座位284bp／284bp、BM1258座位106bp／92bp、BM1443座位166bp／160bp、BM1905座位187bp／187bp、BM302座位142bp／140bp、BM4505座位240bp／236bp和CYP21座位215bp／198bp所对应的体细胞评分最小二乘均值较低,对乳房炎抗性而言它们是各自座位上的最有利基因型;微卫星BM1818座位286bp／286bp、BM1258座位102bp／102bp、BM1443座位170bp／160bp、BM1905座位197bp／195bp、BM302座位154bp／145bp、BM4505座位240bp／238bp和CYP21座位204bp／192bp所对应的体细胞评分最小二乘均值较高,对乳房炎抗性而言它们是各自座位上的最不利基因型。     

Estimation of Mutation Rates Based on the Analysis of Polypeptide Constituents of Cultured Human Lymphoblastoid Cells

A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.     

Evaluating the utility of microsatellites for investigations of autopolyploid taxa

Autopolyploid taxa present numerous challenges for population genetic analyses due to difficulties determining allele dosage. Dosage ambiguity hinders accurate assessment of allele frequencies, multilocus genotypes (MLGTs), as well as levels and patterns of clonality. The pervasiveness of polyploidy in the evolutionary history of plant taxa makes this a recurring problem. Whereas diploidization of loci may occur over time, duplication of at least some loci is still frequently evident. Fortunately, with high-quality allozyme gels, it is possible to accurately infer allele dosage and, thus, determine exact MLGTs. However, accurately assessing dosage of microsatellite peaks is nearly impossible when studying wild populations with a large number of alleles per locus. Even if precise knowledge of genotypes is not required, for comparable numbers of alleles per locus and loci, the number of "phenotypes" is always lower with microsatellites than allozymes due to the inability to assess allele dosage. Microsatellite loci typically have more alleles per locus relative to allozymes although fewer loci are generally employed. Here, we present a mathematical model for comparing the relative utility of simple sequence repeat (SSR) versus allozyme markers to discriminate MLGTs. For example, the average plant allozyme study (2.6 alleles per locus, 10 polymorphic loci) has better discriminating power than SSR markers with 10 alleles at each of 3 loci, 9 alleles at 4 loci, 6 alleles at 5 loci, 5 alleles at 6 loci, and 4 alleles at 8 loci, demonstrating the value of assessing the relative discriminating power of these markers.     

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.     

Mapping, identification of polymorphisms and analysis of allele frequencies in the porcine skeletal muscle myopalladin and titin genes

Genes coding for sarcomeric proteins may play a key role in muscle mass accretion and meat production. Screening a skeletal muscle cDNA library we isolated two partial sequences coding for the sarcomeric myopalladin and titin genes. In the present work we identified three SNPs in the 3' untranslated region, two at the myopalladin locus and one at the titin locus. Myopalladin was mapped on porcine chromosome (SSC) 14 using a somatic cell hybrid panel, a radiation hybrid panel and by linkage mapping. The linkage mapping of titin confirmed the position on SSC15. Then we analysed the allelic distribution of the alleles at both loci in six different porcine breeds. The analysis of the allele frequencies for these two loci in extremely divergent groups of pigs selected according to lean cuts (LC) and average daily gain (ADG) approached the significance level for myopalladin and LC trait. Further studies are needed to test the presence of a putative effect of myopalladin on lean meat content.     

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to chromosome 19.

We have discovered and characterized a compound polymorphic locus on chromosome 19, defined by an arbitrary genomic DNA segment cloned into a cosmid vector. Four different restriction fragment length polymorphisms with minor allele frequencies equal to or greater than 10% are revealed by Southern hybridization of subclones of cosmid 1-13 with TaqI, MspI, BamHI, and HindIII digests of human DNAs. Seventy-two percent of unrelated individuals are heterozygous at one or more loci, and seven of the 24 possible haplotypes occur with frequencies of 3%-38%. Using a somatic cell hybrid panel, we have mapped this locus to 19p13.2----19q13.3, whereas in situ hybridization suggests the probe is on 19p. Taken together, these results suggest localization to 19p13.2----19cen. The locus revealed by probes from cosmid 1-13 has been designated D19S11.     

Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.     

Detecting QTL for feed intake traits and other performance traits in growing pigs in a Piétrain-Large White backcross

Knowing the large difference in daily feed intake (DFI) between Large White (LW) and Piétrain (PI) growing pigs, a backcross (BC) population has been set up to map QTL that could be used in marker assisted selection strategies. LW × PI boars were mated with sows from two LW lines to produce 16 sire families. A total of 717 BC progeny were fed ad libitum from 30 to 108 kg BW using single-place electronic feeders. A genome scan was conducted using genotypes for the halothane gene and 118 microsatellite markers spread on the 18 porcine autosomes. Interval mapping analyses were carried out, assuming different QTL alleles between sire families to account for within breed variability using the QTLMap software. The effects of the halothane genotype and of the dam line on the QTL effect estimates were tested. One QTL for DFI (P < 0.05 at the chromosome-wide (CW) level) and one QTL for feed conversion ratio (P < 0.01 at the CW level) were mapped to chromosomes SSC6 - probably due to the halothane alleles - and SSC7, respectively. Three putative QTL for feed intake traits were detected (P < 0.06 at the CW level) on SSC2, SSC7 and SSC9. QTL on feeding traits had effects in the range of 0.20 phenotypic s.d. The relatively low number of QTL detected for these traits suggests a large QTL allele variability within breeds and/or low effects of individual loci. Significant QTL were detected for traits related to carcass composition on chromosomes SSC6, SSC15 and SSC17, and to meat quality on chromosome SSC6 (P < 0.01 at the genome-wide level). QTL effects for body composition on SSC13 and SSC17 differed according to the LW dam line, which confirmed that QTL alleles were segregating in the LW breed. An epistatic effect involving the halothane locus and a QTL for loin weight on SSC7 was identified, the estimated substitution effects for the QTL differing by 200 g between Nn and NN individuals. The interactions between QTL alleles and genetic background or particular genes suggest further work to validate QTL segregations in the populations where marker assisted selection for the QTL would be applied.