Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

The polymorphism and haplotype of TLR3 gene in grass carp (Ctenopharyngodon idella) and their associations with susceptibility/resistance to grass carp reovirus

Toll-like receptors (TLRs) have emerged as crucial sensors of invading microbes through recognition of pathogen-associated molecular patterns (PAMPs) in viruses, bacteria, fungi and protozoa. The polymorphisms in TLRs are closely associated with the resistance to pathogen infections. TLR3 involved in the recognition of double stranded RNA in humans, mice, pigs and fishes. In present study, the nucleotide sequence polymorphisms of TLR3 gene in grass carp (Ctenopharyngodon idella) (CiTLR3) were investigated to explore their association with susceptibility/resistance to grass carp reovirus (GCRV). Twelve single nucleotide polymorphisms (SNPs) and an ins/del mutation were detected in the complete sequence of CiTLR3. Ten of them were sited in the non-coding region. The two SNPs in exon were synonymous mutation. The ins/del mutation was coincidental at the start codon. To investigate the association between the polymorphism and the susceptibility/resistance to GCRV, we selected eight SNPs in the non-coding region and analyzed the genotype and allele distribution in susceptible and resistant groups with PCR-RFLP. The statistical results indicated that only ?764 G/T was significantly associated with the resistance of grass carp to GCRV both in genotype (P = 0.040) and allele (P = 0.025). Linkage disequilibrium analysis revealed ?543 A/G, ?488 G/T, 4116 G/T and 4731 C/T were linkage disequilibrium, and haplotype analysis revealed that haplotype GTTT frequency in susceptible group was significantly higher than that in the resistant group (OR = 2.01, 95% CI 0.996–4.043, P = 0.049). To further confirm the correlation, an additional infection experiment was carried out. The mortality in the ?764 GG genotype individuals was significantly lower than GT genotype (OR = 0.208, 95% CI 0.067–0.643, P = 0.011) and TT genotype (OR = 0.183, 95% CI 0.052–0.648, P = 0.015). All the results indicated that haplotype GTTT and genotype ?764 TT and ?764 GT individuals were susceptible to GCRV while ?764 GG was resistant, which could be the optional markers for selective breeding for the GCRV-resistant grass carp in future.     

A novel 18-bp deletion mutation of the AMPD1 gene affects carcass traits in Qinchuan cattle

The objectives of the present study were focused on detecting deletion mutation in bovine AMPD1 gene, and analyzing its effect on body measurement and carcass traits in Qinchuan cattle by using DNA sequencing and agarose electrophoresis methods. The 198-bp PCR products of AMPD1 gene exhibited three genotypes and two alleles were revealed: A and B. The frequencies of genotype AA/AB/BB in Qinchuan populations was 0.7163, 0.2233 and 0.0605. The χ²-test analysis demonstrated that the breed was not in agreement with Hardy–Weinberg equilibrium (P < 0.05). The association of the 18-bp deletion mutation of AMPD1 gene with body measurement and carcass traits of Qinchuan cattle were analyzed. The cattle with AA genotype had slaughter weight and carcass weight than those with genotype AB (P < 0.01 or P < 0.05). These results suggest that the 18-bp deletion mutation may influence the carcass traits in Qinchuan cattle.     

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。     

MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus)

In this study, the association between MH-DAB gene polymorphism and disease resistance was evaluated by challenging grass carp (Ctenopharyngodon idellus) with Flavobacterium columnare. Eight genotypes and six alleles were found, and named by common nomenclature. The genotypes AA, BB, EE, and DE, and the alleles Ctid-DAB1*0101, Ctid-DAB1*0201 and Ctid-DAB1*0401 were more preponderant in fish. The genotype BB was associated with higher resistance to F. columnare, as well as two alleles Ctid-DAB*0101 and Ctid-DAB*0201. Allele Ctid-DAB*0102 has decreased resistance to F. columnare. The expression of MH-DAB gene was decreased in the liver, kidney, and intestine but not in the spleen, gill, and skin at 2 days post infection (dpi), versus to that in the control group. MH-DAB gene expression was up-regulated in most tissues but remained at normal levels in the intestine at 15 days post infection. Our data suggested that MH-DAB polymorphism can be used as a potential genetic marker for disease resistance breeding of grass carp in the future.     

The CSN1S1?N and F alleles identified by PCR-SSCP and their associations with milk yield and composition in Chinese dairy goats

A method was depicted to identify null allele CSN1S1 N and low allele CSN1S1 F of the CSN1S1 gene of goat using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism). First, primer A was designed to amplify the exon 9 of CSN1S1 gene which produced three genotypes AA, AB, and BB. Among these three genotypes, only AA and AB individuals had a cytosine deletion at exon 9 after DNA sequencing, which cannot be used to identify the N and F alleles. Therefore, primer B was used to amplify intron 14 of CSN1S1 of described AA and AB individuals. Genotypes FF, FN and NN were detected within AA individuals and genotypes FO and NO were detected in the above AB individuals. The frequencies of F and N alleles in 708 samples from Xinong Saanen (XS) and Guanzhong (GZ) dairy goat breeds were 0.1139, 0.0927, and 0.2376, 0.1193, respectively. In 268 XS samples, the individuals with NN genotype contained a significant lower protein content than that of other genotypes (P<0.01). Individuals of FF genotype had significant higher milk yield than that of NO genotype in the first milk lactation of 202 XS individuals (P<0.05). Therefore, the variability at CSN1S1 locus contains enough genetic diversity to be potentially useful in improving the quality and production of milk in Chinese dairy goat breeds.     

Meta-analysis of Genetic Structure and Association of Prolactin Gene with Performance Traits in Dairy Cattle in India

The present meta-analysis was carried to provide the more reliable estimates of gene frequency and association of Rsa 1 generated candidate genotype of prolactin gene within exon-3 with performance traits in 1198 Indian dairy cows using data from 15 published studies. Six genetic models viz., codominant (AA vs. AB, AA vs. BB and AB vs. BB), dominant (AA+AB vs. BB), completely over dominant (AA+BB vs. AB) and recessive (AA vs. AB+BB) were used to obtain standardized mean difference (SMD) between genotypes. Meta-analysis showed that the gene frequency of A allele (156 bp) was 0.60 (95% confidence interval (CI) 0.54, 0.65). In association analysis, cows with AB genotype [SMD?=?0.65, 95% CI 0.00, 1.30] had significantly (P?<?0.05) higher lactation milk yield (LMY) as compared to BB genotype, whereas AA and AB genotypes had similar trend. Likewise, AA?+?AB also had larger effect [SMD?=?2.31, 95% CI 0.21, 4.10] on LMY as compared to BB. Cows with AB genotype had significantly lower age at first calving (AFC) with small effect [SMD (AA vs. AB)?=?1.38, 95% CI 0.06, 2.70] and medium effect [SMD (AB vs. BB)?=????3.83, 95% CI???6.41,???1.24] as compared to cows with AA and BB genotypes, respectively. This finding was confirmed under dominant and completely over dominant models. In case of fat%, AA genotype showed negative effect (SMD?=????0.51, 95% CI???0.84,???0.17) under recessive model. It was concluded that the propagation of allele A is promising to help dairy farmers to improve the genetic quality of their dairy cows.

     

南方养殖草鱼呼肠孤病毒的分子特性比较及双重PCR检测方法的建立

本实验室2009年从广东省患典型出血病的养殖草鱼中分离到一株具强致病性的水生呼肠孤病毒GCRV-GD108株,该毒株具有11个节段双链RNA。全基因组序列分析显示与草鱼呼肠孤病毒GCRV及水生呼肠孤病毒属其它已知种存在较大的分子差异。本研究进一步检测了广东、福建、湖南等地草鱼出血病流行毒株的分子特性。根据已克隆到的GCRV-GD108株11个节段序列分别设计合成特异引物,从各地收集患出血病草鱼,提取组织总RNA,RT-PCR检测。结果表明各检测样品均可扩增到特异性条带,而GCRV标准株则无特异条带;同时根据GCRV标准株序列合成的特异引物进行扩增,GCRV标准株有特异性条带,而各检测样品则均无带。测序结果显示各样品间相应片段序列的同源性很高(95.2%~99.4%),与GCRV-GD108的相应序列也具高同源性(95.0%~99.8%),说明检测样品与GCRV-GD108株具有相似的分子特性,均与GCRV及水生呼肠孤病毒属的其它种存在较大差异。本研究结果提示我国养殖草鱼出血病病毒存在着不同的分子类型,GCRV-GD108株在南方具有一定的代表性,在病害防控上尤其是疫苗研制与使用上应予关注。此外,从上述引物中筛选出适合于双重PCR的引物对,建立了双重PCR检测方法,可在一次PCR反应中鉴别所感染的病毒属于GCRV或GCRV-GD108株。     

西藏牦牛ADD1基因第2外显子的PCR-SSCP检测及序列分析

利用PCR-SSCP和DNA测序方法对帕里牦牛、工布江达牦牛、类乌齐牦牛、康布牦牛、桑日牦牛、巴青牦牛、江达牦牛、斯布牦牛、嘉黎牦牛、桑桑牦牛、丁青牦牛等西藏11个牦牛类群共483头牦牛的ADD1基因(被认为是极可能影响肉质的候选基因)第2外显子序列进行遗传多态性分析。结果表明,66 bp处碱基T缺失和256 bp处A→G突变,共有AA、AB、BB 3种基因型;其中帕里牦牛只有AA基因型,江达牦牛只有AB基因型,康布牦牛、嘉黎牦牛、巴青牦牛、桑日牦牛、斯布牦牛均缺少BB基因型,丁青牦牛、类乌齐牦牛缺少AB型,均存在严重偏态;桑桑牦牛、工布江达牦牛中存在AA、AB、BB 3种基因型。除江达牦牛外,其它10个牦牛类群中AA为优势基因型,A基因为优势等位基因,分布较高。除帕里牦牛、巴青牦牛外,其它9个牦牛类群均处于中度多态(0.25相似文献   

IGFBP3基因多态性与秦川牛部分屠宰性状的相关性 Polymorphisms of Insulin-like Growth Factor Binding Protein 3 Gene and Its Associations with Several Carcass Traits in Qinchuan Cattle

以IGFBP3基因作为秦川牛（Bos taurus）部分屠宰指标的侯选基因,在对60头秦川牛的IGFBP3基因进行PCR-RFLP和序列分析的基础上,对秦川牛群体中IGFBP3基因座等位基因和基因型频率的分布及其与秦川牛部分屠宰性状的关系进行了分析。结果发现,在秦川牛群体中,651 bp的PCR 产物经过限制性内切酶HaeIII消化后,表现出3种基因型,其中等位基因A、B及3种基因型AA、AB、BB的频率分别为0.84、0.16和070、0.28、0.02。经序列分析发现,第299位的C→A颠换（GGCC变成了GGAC）导致了1个HaeIII限制性酶切位点的丢失而产生了该基因座多态性。在所研究的群体中,该多态基因座处于Hardy-Weinberg平衡状态（P＞005）。对13头24月龄秦川牛进行屠宰分析,发现不同基因型对秦川牛部分屠宰指标有一定影响,AA、AB及BB型个体的屠宰率、净肉率及西冷、牛柳、眼肉和嫩肩肉的产率逐渐降低,但差异不显著（P＞0.05）;AA型个体的眼肌面积大于BB型个体（P＜0.05）,AB型和BB型个体胴体脂肪含量高于AA型个体（P＜0.01）。 Abstract:DNA samples from 60 Qinchuan cattle (Bos taurus) were analyzed with PCR-RFLPs and sequencing for insulin-like growth factor binding protein 3 (IGFBP3) gene.Fragments of 651 bp were amplified with two primers and the products of PCR were digested with restriction endonuclease HaeIII.The produced fragments showed three genotypes,namely AA,AB and BB after electrophoresis.Frequencies of the genotype AA,AB,BB and allele A,B were 0.7,0.28,0.02,and 0.84,0.16,respectively.Sequence analysis showed that a transversion of C→A at 299 nt resulted in loss of the cleaved site of restriction endonuclease HaeIII and produced this polymorphism.This polymorphic locus of IGFBP3 gene was at Hardy-Weinberg equilibrium (P＞0.05).The genotypes of AA,AB,BB slightly affected several slaughter and carcass traits of Qinchuan cattle.Dressing percentage,net meat percentage,striplion percentage,tenderloin percentage,ribeye percentage and tender shoulder percentage were decreased with the genotypes of AA,AB and BB in Qinchuan cattle,but it was not significant (P＞0.05).Average ribeye area in individuals of AA genotype was significantly higher than that in individuals of BB genotype (P＜0.05),and beef fat content in individuals of genotype AB and BB was significantly higher than that in individuals of AA genotype (P＜0.01).     

Polymorphism of bone morphogenetic protein 4 gene and its relationship with litter size of Jining Grey goats

Two pairs of primers (P1 and P2) were designed to detect single nucleotide polymorphisms of exon 2 and intron 2 of bone morphogenetic protein 4 (BMP4) gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Boer, Angora and Inner Mongolia Cashmere goats) by single strand conformation polymorphism. Results showed that no polymorphism was detected for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2 (primer P2), three genotypes (AA, AB and BB) were detected in Jining Grey and Inner Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats, and only one genotype (AA) in Boer goats. Sequencing revealed one mutation (2203G>A) of BMP4 gene in the genotype BB in comparison to the genotype AA. The differences of litter size between AA, AB and BB genotypes were not significant (P > 0.05) in Jining Grey goats. A pair of primer (P3) was designed to detect polymorphism in the 3' flanking region of BMP4 gene that contained dinucleotide repeated sequence (CA) in the four goat breeds by microsatellite analysis. For primer P3, three genotypes (CC, CD and DD) were detected in four goat breeds. Sequencing revealed one more CA dinucleotide in genotype DD than in genotype CC. The Jining Grey does with genotype CC had 0.55 (P < 0.05) or 0.72 (P < 0.05) kids more than those with genotype CD or DD. These results preliminarily indicated that allele C of BMP4 gene is a potential DNA marker for improving litter size in goats.     

IGFBP-3基因多态性及其与中国美利奴羊部分羊毛性状的关联性分析

采用PCR-SSCP方法对中国美利奴羊和哈萨克羊中IGFBP-3基因的多态性进行了检测, 并对不同基因型与中国美利奴羊部分羊毛性状间的关联性进行了分析。结果在位于内含子1区的一段178 bp的扩增产物经SSCP分析后出现了3种基因型, 基因型AA、AB和BB及等位基因A、B在中国美利奴羊中的频率分别为0.70、0.24、0.06和0.82、0.18; 在哈萨克羊中的频率分别为0.87、0.13、0.00和0.93、0.07。序列分析发现: 在该序列的122位碱基表现多态性 (g.122 G>T)。所研究的两个群体在该位点上均处于Hardy-Weinberg不平衡状态(P<0.01)。不同基因型对部分羊毛性状有一定的影响: 不同基因型个体在剪毛后体重和净毛率上没有明显差异。AA、AB及BB基因型个体的羊毛伸直长度逐渐变短, 其中AA与AB基因型之间差异极显著(P<0.01)。AA型个体的剪毛量和羊毛密度要明显低于AB型(P<0.01)和BB型个体(P<0.05); 羊毛纤维直径则明显高于AB型(P<0.01)和BB型(P<0.05)个体。     

荷斯坦牛Nramp1基因遗传多态性及其与乳房炎相关性的研究

利用PCR-SSCP技术检测了344头中国荷斯坦牛Nramp1基因exon 11的基因多态性, 并分析了其不同基因型与乳房炎及产奶量性状的关系。结果表明: 实验群体发现3种基因型AA、AB、BB, 其中A等位基因为优势等位基因, 等位基因频率为0.767, 而B等位基因频率则为0.233。经χ2适合性检验, 群体处于Hardy-Weinberg平衡状态(P>0.05)。测序结果显示: 扩增片段分别在200 bp(C/G)和254 bp(T/G)存在碱基突变, 并导致了氨基酸改变, 分别为丙氨酸替换为脯氨酸(Ala356Pro)、亮氨基酸替换为蛋氨酸(Leu374Met)。通过构建最小二乘线性模型, 进行Nramp1基因多态性与产奶量、体细胞评分(SCS)的相关性分析表明, AA型个体的SCS最小二乘均值显著低于BB﹑AB型(P<0.05), 而AA型﹑AB个体的产奶量最小二乘均值显著高于BB型(P<0.01, P<0.05), AA基因型可作为乳房炎抗性的优良基因型。因此, 可将Nramp1作为奶牛乳房炎候选基因应用于分子标记辅助选择育种。     

FSHβ 基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究

采用PCR-SSCP技术检测促卵泡素b亚基(follicle-stimulating hormone β, FSHβ)基因5′调控区、外显子1和外显子2在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊、安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 山羊与绵羊的FSHβ 基因该段核苷酸序列同源性为98%。9对引物中, 只有P9的扩增片段存在多态性。P9的扩增片段在济宁青山羊和辽宁绒山羊中检测到AA、AB和AC 3种基因型; 在波尔山羊中检测到AA、CC和AC 3种基因型; 在安哥拉山羊中检测到AA、BB、CC、AB、AC和BC共6种基因型。测序分析发现BB型与AA型相比在外显子2的第94 bp处有G→A突变, 并引起氨基酸改变(丙氨酸→苏氨酸); CC型与AA型相比在外显子2的第174 bp有一处C→T沉默突变。济宁青山羊AA、AB和AC基因型频率分别为0.686、0.137和0.177。AA基因型济宁青山羊产羔数最小二乘均值比AB基因型的多0.78只(P<0.05), 比AC基因型的多0.64只(P<0.05)。     

猪IGF-I基因的一个遗传多态性及其遗传效应分析

采用PCR-SSCP方法对长白猪（87头）、大白猪（79头）和马身猪（102头）的胰岛素样生长因子-I（IGF-I）基因exon3和exon4分别进行单核苷酸多态性分析。发现exon3上有多态性,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,多重比较差异极显著（P<0.01）。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不显著（P>0.05）。最小二乘分析结果表明,BB基因型与其它2种基因型比较有较大的初生重,同AA和AB型比较差异极显著（P<0.01）,3种基因型在初生重的大小排列顺序为AA相似文献