猪CAPN1基因部分外显子及3''UTR区的SNPs检测

以野猪.民猪和大白猪为研究对象,根据网上公布的序列设计了7对引物,采用测序,PCR-SSCP和PCR-RFLP方法对CAPN1基因的部分外显子和3'UTR区进行了单核苷酸多态性检测和基因型分析,探讨CAPN1基因多态性与瘦肉率和嫩度的关系.研究发现11个SNPs,其中5个位于外显子,4个位于内含子,2个位于3'UTR区,外显子中的突变有一处是错义突变,导致了蛋白质多肽链第260位氨基酸发生了M/V的替代.群体遗传学分析表明,在所检测的各多态位点上,野猪、民猪、大白猪3个品种间不同基因型的分布都存在着极显著的差异(P<0.01),而野猪和民猪之间各基因型的分布差异不显著(P>0.05),民猪和大白猪之间各基因型的分布存在着极显著的差异(P<0.01).结合品种特性分析表明,P4、P6引物和3'UTR区Hinf1位点所检测的不同基因型和瘦肉率具有一定的相关性.     

野猪、民猪、大白猪μ-钙激活酶基因的变异位点分析

为了进一步研究CAPN1基因变异与肉嫩度的关系, 寻找与猪嫩度性状相关的分子标记, 对CAPN1基因组进行了克隆、测序, 并利用PCR-SSCP方法对其编码区序列进行了分子扫描, 寻找多态位点, 分析不同基因型在野猪、民猪、大白猪中的种间分布规律。获得了猪CAPN1基因的15个内含子序列; 根据GenBank上提供的CAPN1 CDS及克隆的内含子序列设计了5对多态性引物进行PCR-SSCP分析; 共找到8个SNPs, 其中7个位于外显子上, 1个位于内含子上, 并且外显子上的突变有3个是错义突变, 分别造成了蛋白质多肽链上第54位氨基酸的S/T、第192位氨基酸的G/E、第363位氨基酸的V/I替代; χ²独立性检验表明不同基因型在大白猪与野猪、民猪之间存在着极显著的差异(P<0.01), 野猪和民猪之间除了S1引物3种基因型的分布存在显著差异外(0.010.05)。这些多态位点具有成为分子标记的潜在可能。     

野猪MC4R基因的克隆及变异初步研究

黑素皮质素受体4是在人类肥胖研究中发现的重要调节因子,参与调节动物的体重、采食量和能量稳态,缺失MC4R基因的突变纯合体小鼠出现遗传性肥胖。为了进一步揭示其群体遗传变异,寻找新的遗传标记,本研究对野猪(Sus scrofa ussuricus)MC4R基因进行了克隆(GenBank accession NoDQ388767)和序列分析,并对所发现的错义突变进行了基于限制性内切酶HindⅢ的PCR-RFLP分析。序列分析表明野猪与民猪MC4R基因的编码区序列完全相同,与大白猪相比存在4个SNPs;对14头野猪的酶切多态性分析表明该突变位点是多态位点,并且3种基因型的分布符合Hardy-Weinberg定律。结果表明,野猪具有独特的遗传信息。     

猪TLR4基因外显子1新等位基因的分离及遗传变异分析

文章采用PCR-SSCP方法对亚洲野猪、3个引进的商业化品种和10个中国地方猪品种共893个个体TLR4基因外显子1的遗传变异进行了检测,旨在系统分析国内外猪种TLR4基因的多态性,为探讨该基因在免疫和防御系统中发挥的作用提供依据。结果,在猪TLR4基因外显子1中分离到新的等位基因,共检测到3个等位基因,6种基因型。其中杜洛克检测到AA、BB、CC、AB、AC、BC基因型,有杜洛克血统的苏太猪中检测到BB、CC、BC基因型,长白猪、约克夏中检测到CC、BC基因型,野猪及所有10个中国地方猪品种TLR4基因外显子1高度保守,只检测到CC基因型,中国地方猪品种和引进品种TLR4基因外显子1多态性存在极显著的差异。3种基因型中CC型与GenBank中的序列一致,BB和AA基因型分别存在G93C同义突变位点和G194A无义突变位点,这2个变异位点与抗逆性和一般抗病力的关系值得进一步深入研究。     

猪整合素β1基因CRS-PCR多态性与产仔数的关联性分析

文章采用CRS-RFLP技术对长白猪、大白猪和杜洛克猪3个品种的整合素β1基因第5外显子T32207C位点及第7外显子A35230G位点进行单核苷酸多态性分析,并将基因多态性与猪的产仔数进行关联分析。结果表明:32207多态位点的基因型效应对3个品种的总产仔数(TNB)和产活仔数(NBA)影响均不显著;35230多态位点的基因型效应对大白猪和长白猪头胎、二胎及所有胎次的TNB和NBA的影响达到显著(P<0.05)或者极显著水平(P<0.01),基因型GG、AG与AA对产仔数的影响存在差异,其效应为GG,AG>AA。可见整合素β1基因35230位点的G等位基因对大白猪和长白猪的产仔数性状有显著影响。     

野猪CAPN7基因的克隆、表达和变异分析

钙蛋白酶是细胞质中主要的蛋白水解酶,在肌肉生长、蛋白转化及嫩化过程中发挥复杂的作用。本研究利用RT-PCR、生物信息学方法克隆野猪（Sus scrofa ussuricus）CAPN7 cDNA并进行序列分析,利用相对定量RT-PCR方法研究其组织表达情况和利用PCR-SSCP方法对其进行变异分析。结果表明,野猪CAPN7基因编码区全长2 442 bp,预期编码813个氨基酸残基,多肽链中存在着calpain家族的催化结构域和催化活性中心;野猪CAPN7不同程度地表达于所检测的12种组织中,在6、9月龄野家杂交猪肌肉组织中的相对表达量高于同一时期的大白猪肌肉组织;所检测到的3种基因型在野猪、民猪和杜洛克中的分布存在着极显著差异。本研究为进一步揭示calpain7的功能提供了分子生物学基础。     

可乐猪ApoA5基因多态性及其对肉质性状的遗传效应分析

根据GenBank中报道的猪apoA5基因序列设计2对引物,应用PCR技术从可乐猪肌肉组织基因组中扩增出了apoA5基因第三外显子的特异片段,采用直接测序法对apoA5基因进行单核甘酸多态性检测,并分析该基因与可乐猪12项肉质性状的关联性,结果表明:在可乐猪apoA5基因第三外显子上共检测到3个SNPs-突变位点:A~(226)G、G~(535)C、和C~(1514)T,其中A~(226)G和G~(535)C突变发生在编码区内,没有引起氨基酸的改变,为沉默突变。C~(1514)T突变发生在3'-UTR,χ~2检验表明,发现的3个多态位点均处于Hardy-Weinberg平衡状态(p0.05)。A~(226)G位点为低度多态,其余两个突变位点均为中度多态。最小二乘分析显示,apoA5基因第三外显子226位点AA基因型个体的肌内脂肪显著高于AG基因型个体(p0.05),535位点GG基因型个体的肉色显著高于CC和GC型个体(p0.05),1514多态位点与可乐猪肉质性状没有显著关系(p0.05)。     

猪激素敏感脂肪酶基因外显子1的遗传多态性分析

采用PCR-SSCP方法检测猪激素敏感脂肪酶（hormone sensitive lipase,HSL）基因外显子1的多态性,并分析其与初生重、断奶重、6月龄重和背膘厚的关联性。根据猪HSL基因的DNA序列（AJ000483）设计5对引物,结果在P5引物对扩增的片段上发现了多态性,并对纯合子进行测序,发现外显子1的874bp处存在G-A转换,且存在3种基因型（AA、AB、BB）。统计结果表明,3种基因型在各品种中的分布不一致,长白猪和大白猪与莱芜猪和沂蒙黑猪比较差异极显著（P〈0．01）,长白猪与大白猪比较,莱芜猪与沂蒙黑猪比较差异均不显著（P〉0．05）。固定效应模型分析结果表明,背膘厚基因型间差异显著（P〈0．05）,而初生重、断奶重和6月龄重基因型间差异均不显著（P〉0．05）。最小二乘分析结果表明,BB基因型个体同AB和AA基因型个体比较背膘厚的差异显著（P〈0．05）,3种基因型在背膘厚的大小排列顺序为BB〈AB〈AA。因此,推测HSL基因对个体胴体瘦肉率存在一定的影响将HSL某因应用于猪育种过程中的标记辅助选择可以加快猪的育种进程。     

鸡Myostatin基因单核苷酸多态性的群体遗传学分析

肌肉生长抑制素是控制骨骼肌生长发育的重要细胞因子,采用PCR－SSCP和测序的方法发现了5个位于Myostatin基因5′－和3′－调控区的单核苷酸多态性位点,对北京油鸡、白耳鸡、石歧杂、矮小黄鸡、小型黄鸡、惠阳胡须鸡、隐性白羽鸡、海兰、AA鸡等不同鸡种的该单核苷酸多态性分析结果表明：Myostatin基因的5′调控区引物P60／P61扩增片段多态性是由3个核苷酸的改变而产生的[分别是G→A（304位）、A→G（322位）、G→（344位）],引物P93／P94扩增片段的多态性是由G→A（167位）突变造成的,引物P117。P118PC扩增片段多态性是由T→C（177位）造成的。3′调控我引物P80／P81扩增片段多态性是由第7263位A突变为T造成的,引物P76／P77扩增片段多态性是由A→G（6935位）造成的。不同鸡种群体遗传学分析表明,5′－调控区引物60／P61扩增片段多态性片段多态性是由A→G（6935位）造成的。不同鸡种群体遗传学分析表明,5′－调控区引物P60／P61扩增片段多态性位点在北京油鸡的基因型频率分布与其他的品种有很大的差异,其BB型频率为0．700,AA基因型频率仅为0．033,而其他鸡种中以A基因优势;对于引物P93／P94,品种间的基因型频率差异极显著（P＜0．01）,北京油鸡和AA鸡的EE型频率鸡种中以A基因占优势;对于引物P93／P94,品种间的基因型频率差异极显著（P＜0．01）,北京油鸡和AA鸡的EE型频率低于其他品种,白耳鸡和海兰蛋鸡以EE型为主,其频率高于其他品种;3′－调控区引物P80／P81多态怀位点在9个鸡种中都是等位基因C占优势。引物P76／P77,总体上MM型的频率较低,杂合子MN型的频率较高。     

猪IGF2基因外显子4b的遗传多态性及其遗传效应

采用PCR-SSCP方法检测猪胰岛素样生长因子2(insulin-like growth factor 2,IGF2)基因外显子4b的多态性,并分析其对初生重、断奶重、6月龄重和背膘厚的遗传效应。根据猪IGF2基因的DNA序列(AY044828)设计引物,在exon4b上发现了一个多态性位点,并对纯合子进行测序,发现13位C→A转换,且存在3种基因型(AA、AB、BB)。统计结果表明,3种基因型在各品种中的分布不一致,长白猪与大白猪比较,莱芜猪与大薄莲猪比较,沂蒙黑猪和里岔黑猪比较差异不显著(P>0.05);其他猪种间基因型分布的差异均显著(P<0.05)。固定效应模型分析结果表明,初生重,断奶重和背膘厚基因型间差异显著(P<0.05),而6月龄重基因型间差异不显著(P>0.05)。最小二乘分析结果表明,BB基因型个体同AA和AB、基因型个体比较初生重和断奶重的差异显著(P<0.05),3种基因型在初生重和断奶重的大小排列顺序为AA>AB>BB;AA基因型个体同AB和BB基因型个体比较背膘厚的差异显著(P<0.05),3种基因型在背膘厚的大小排列顺序为BB>AB>AA。因此,推测IGF2基因对个体的生长速度和胴体瘦肉率存在一定的影响,将IGF2基因应用于猪育种过程中的标记辅助选择可以加快猪的育种进程。     

猪Toll样受体4基因SNPs功能分析

Toll样受体4(Toll-like receptor 4,TLR4)在机体的免疫反应中发挥重要作用,该基因突变会影响受体的信号转导能力和机体的疾病抗性/易感性。文章在前期工作的基础上,进一步分析c.611 T>A(p.Leu204His)、c.1027C>A(p.Gln343Lys)和c.1605 G>T(p.Leu535Phe)3个错义突变对猪TLR4功能的影响。利用RT-PCR方法克隆猪TLR4基因全长编码区并引入定点突变;利用真核表达、双荧光素酶报告系统和Western blotting方法在瞬时转染的PK-15细胞内研究3个单核苷酸多态(Single nucleotide polymorphisms,SNPs)对猪TLR4配体识别和信号转导能力的影响;同时,利用创造酶切位点PCR-RFLP方法分析对TLR4活性有显著影响的点突变在民猪、大白、长白和中国东北野猪4个群体中的分布。结果,成功获得了民猪TLR4基因的全长编码区和3个单碱基变异体,构建了不同等位基因的真核表达载体,在PK-15细胞内确定了c.1605 G>T变异导致TLR4向下游传递信号的能力显著降低(P<0.01),该SNP只存在于民猪和野猪中且频率较高。猪TLR4基因c.1605 G>T变异影响Toll样受体的信号传递,可能和机体的疾病抗性/易感性有关。     

三江白猪与其他5个品种猪的群体遗传关系的分析

应用100条RAPD引物对三江白猪及其杂交亲本长白猪和东北民猪,以及大约克夏、圣特西和法系大白猪进行亲缘关系的DNA多态分析。其中15条引物的扩增结果具有明显的多态,将其扩增结果进行聚类分析,发现三江白猪与长白猪(遗传距离d=0.090)、与东北民猪(d=0.175)的亲缘关系较近,与大约克夏(d=0.263)、法系大白(d=0.223)、圣特西猪(d=0.580)的亲缘关系较远。     

Quantitative trait loci for carcass traits on pig chromosomes 4, 6, 7, 8 and 13

For 22 carcass traits, we identified 16 QTLs (based on data for pig resource population no. 214, including 180 F2 hybrids of 3 Yorkshire boars and 8 Meishan sows) and mapped them with the use of 39 microsatellite marker loci on chromosomes 4, 6, 7, 8 and 13. Five QTLs were highly significant (P < or = 0.01 at chromosome level): for skin weight (on chromosome 7 at SW1856 and on chromosome 13 at SW1495), skin percentage (on chromosome 7 between SW2155 and SW1856 and on chromosome 13 between SW1495 and SW520), and ratio of leg and butt to carcass (on chromosome 4 at SW1996). The remaining 11 QTLs were significant (P < or = 0.05 at chromosome level): for backfat thickness at shoulder, loin eye width, loin eye height, fat meat weight, lean meat weight, skin weight, bone weight, skin percentage, fat meat percentage, and ratio of lean meat to fat meat. The proportion of phenotypic variance explained by these QTLs ranged from 0.06% (QTL for loin eye width on chromosome 8 between SW1037 and SW1953) to 18.04% (QTL for ratio of lean meat to fat meat on chromosome 7 between SW252 and SW581). Seven of the QTLs reported here are novel.     

Olfactory evaluation of boar taint: effect of factors measured at slaughter and link with boar taint compounds

There is a commitment by the European pig sector to ban surgical castration of male piglets in the European Union in 2018. One alternative to castration is to raise entire male pigs, with an increased risk of boar taint. A field study was performed to: (1) evaluate inter- and intra-farm variation in boar taint prevalence, (2) investigate factors measured at slaughter influencing boar taint and (3) evaluate the relationship between sensorial scoring by a trained panel and the concentration of boar taint components. From 34 farms, neck fat samples were collected from all entire male pigs in at least two slaughter batches per farm (78 batches; 9167 animals). In addition to olfactory boar taint analysis, data were also collected on fresh skin lesions (score 0 to 3) at the slaughter line, slaughter weight, lean meat percentage, duration of transport, time spent in lairage, total delivery duration, day length, shortening of days and outdoor mean temperature. Using the hot iron method, neck fat samples were scored (eight-point scale) for boar taint. Average boar taint prevalence (score ≥3) was 5.6±2.5% and the mean difference between the maximum and minimum prevalence per farm was 4.3±3.2%. Androstenone (AND), skatole (SKA) and indole concentrations were measured for a subset (n=254) of the samples. According to binomial univariate mixed models, entire male pigs with a higher skin lesion score had higher odds of having boar taint (P=0.031), as did fatter entire male pigs (P<0.001). In the binomial multivariate mixed model lean meat percentage (P<0.001) and outdoor mean temperature (P=0.005) remained as only significant factors. Based on our results, we can conclude that these statistically significant at least partially influence the prevalence of boar taint. According to the binomial univariate mixed models SKA concentration in liquid fat seems a better predictor for boar taint than AND. There were no significant synergetic effects between boar taint compounds.     

猪ACTA2基因的克隆、表达分析及其与生产性状的关联

为鉴定对猪生产性状有重要影响的新分子标记,文章采用电子克隆结合PCR方法获得了猪肌动蛋白α2(Actin alpha 2, ACTA2)基因编码区序列和部分基因组序列,建立了第2内含子C1554T替换的PCR-HinfⅠ- RFLP基因分型方法,在所检测的7个不同猪群中除大白和梅大群体外,其他群体中均是C等位基因频率高于T等位基因的频率。标记与性状关联分析发现ACTA2基因型与肩部背膘厚、臀部背膘厚、肥肉率、瘦肉率、股二头肌pH和肌内脂肪显著或极显著相关,TT基因型与CC基因型相比具有更高的瘦肉率以及更低的肥肉率和背膘厚。通过Real-time RT-PCR分析发现ACTA2在大白和梅山两个品种猪骨骼肌中的表达量都随着日龄的增加而降低,在各个阶段,梅山猪中的表达量都比大白猪中的表达量高。     

Reconstruction of the origin of modern swine breeds by mitochondrial gene polymorphism

Mitochondrial DNAs of six pig breeds of Euro-American origin, one breed of Asian origin and of wild boar were studied using PCR-RFLP analysis to detect probable maternal lines of Italian origin among modern pig breeds. In the studied animals the Italian Wild boar haplotype characterized by single nucleotide substitution in the 15524 position of pig mitohondrial genome has not been detected.     

Multiple Asian pig origins revealed through genomic analyses

Previous mitochondrial DNA (mtDNA) studies have suggested that European and Asian pig populations were derived through multiple domestication events. We investigated whether domestic pig populations were derived from distinct ancestors within their respective regions, using eight domestic breeds (five European and three Asian), and also European and Asian wild boar populations. Genomic analyses utilized 21 microsatellite markers (MS) selected for their distribution across the pig genome in addition to the mtDNA D-loop region. The number of alleles per MS loci ranged from 8 (Sw2008) to 16 (S0097 and S0218). Few significant departures from Hardy–Weinberg equilibrium were detected, suggesting the absence of heterozygote deficiencies. Analyses within populations revealed observed mean heterozygosity from 0.48 (Erhualian) to 0.68 (Dutch WB) and an expected mean heterozygosity from 0.53 (Hampshire) to 0.80 (Japanese WB) with effective alleles ranging from 2.28 (Hampshire) to 3.74 (French WB). Wild boar populations demonstrated a higher level of heterozygosity than domestic breeds. Genetic differentiation estimated by fixation indices (F_ST) ranged from 0.021 (Yorkshire and Duroc) to 0.410 (Meishan and Hampshire) and was consistent with previous mtDNA analysis. Both phylogenetic and principal component analyses revealed a distinct separation of European and Asian derived populations with tight clustering of the European domestic breeds. Conversely, the use of both MS and mtDNA clarified that the Asian populations were comprised of three groups, one represented by Erhualian and Meishan breed, the second represented by Lanyu pigs and the third represented by the Asian wild boars. The current findings support the hypothesis that Asian domestic populations were derived from multiple Asian ancestral origins whereas the European domestic populations represent a single ancestral European lineage.