羊RBP4基因DNA池测序分析

选择黔北麻羊、内蒙古白绒山羊、关中奶山羊3个山羊品种构建品种DNA池。对RBP4基因部分CDS区和3’UTR进行PCR产物扩增,并采用直接测序法进行测序。结果表明：所选山羊品种RBP4基因分CDS区和3’UTR区,无突变位点存在。序列分析表明,山羊与其他物种RBP4基因的同源性很高,其中以牛最高,达到98．8％。系统进化分析表明,羊和牛在同一进化支上。生物信息分析表明：RBP4基因所表达蛋白氨基酸序列中有5个磷酸化位点。因此,山羊RBP4基因在进化过程中是高度保守的。研究结果可为进一步分析山羊RBP4基因功能遗传变异提供借鉴。     

贵州地方猪脂肪特异蛋白27基因SNPs筛查与生物信息学分析

以3个贵州地方猪种(可乐猪、贵州白香猪、黔北黑猪)为试验素材构建品种DNA池,采用直接测序技术对猪脂肪特异蛋白27(fat-specific protein 27,Fsp27)基因的第4~5外显子区域进行SNPs快速筛查,共检测出4个SNPs位点:intron3-T2169C,exon4-G5A,intron4-G21C和exon5-C5G,其中exon4-G5A和exon5-C5G使编码氨基酸发生Arg→Gln,Thr→Ser的改变。进一步的生物信息学分析显示,exon4-G5A位点的变异导致mRNA的二级结构改变,exon5-C5G多态位点使蛋白质的二级结构发生变化,且当exon4-G5A和exon5-C5G位点的碱基分别为A和G时蛋白质三级结构与其它3种碱基组合(G与G,G与C,A与C)的三级结构明显不同。     

猪DAZ基因的DNA池测序分析

选取大约克猪、白香猪、可乐猪3个猪种的公猪构建品种DNA池。对DAZ基因的部分CDS区和3'UTR进行PCR扩增,并对PCR产物进行测序。结果表明,3个猪种中,DAZ基因的该序列未发现任何突变。同源性分析结果表明,猪与人、猩猩、猴、鼠、牛的DAZ基因该序列核苷酸同源性分别为97.0%、95.2%、96.1%、92.9%、97.0%。说明在哺乳动物中,其DAZ基因CDS部分序列和3'UTR是高度保守的。系统进化分析结果表明,猪和牛在一个进化支上。蛋白疏水性分析表明,在17－20位有个强疏水区。     

利用DNA池对3个山羊品种CXCL10基因多态性的研究

为了研究3个山羊品种(黔北麻羊、内蒙古绒山羊、关东奶山羊)CXCL10基因多态性,试验采用构建DNA池并直接对其测序的方法进行分析。结果表明:快速筛查到2个与繁殖性能相关的单核苷酸多态性(T-131-C,G-661-A)。进一步利用测序图中的SNP等位基因峰高的比值估算各山羊品种等位基因的频率。     

西藏绒山羊KAP6.2基因SNPs位点的检测及分析

旨在研究绒毛品质相关基因KAP6.2在西藏绒山羊群体中的多态性,分析KAP6.2在不同山羊中的差异。采用聚合酶链反应-单链构象多态性和DNA测序等技术对随机选取的127只西藏绒山羊的 KAP6.2 基因进行突变位点检测及多态性指标分析。结果表明,西藏绒山羊 KAP6.2 基因存在两种基因型 AA和AB,其中等位基因A 为优势等位基因,多态信息含量为0.196 (属低度多态)。经测序并与GenBank中山羊KAP 6.2(No.AY316158)基因序列比对后共发现4处变异,分别为:178nt处24个碱基的缺失导致Ser48>Gly55氨基酸的丢失;113 nt处GT>CC突变导致氨基酸P.19Ser>Trp;121 nt处A>G突变为同义突变;168nt处C>T突变导致氨基酸P.41Thr>Tyr。这些氨基酸的突变很可能导致结构蛋白功能的改变,进而对西藏绒山羊的绒毛品质产生影响。通过研究影响西藏绒山羊绒毛品质的 KAP6.2 基因的多态性,深入了解其分子遗传基础,有利于利用分子遗传标记技术进行西藏绒山羊的选育。     

细毛羊LAMB1基因外显子多态性及其与羊毛纤维直径的关联性分析

试验旨在研究LAMB1基因外显子在细毛羊中的多态性及其与羊毛纤维直径的关联性。基于DNA池重测序技术获得的SNPs数据,共筛选LAMB1基因外显子区域20个SNPs,利用直接测序法、PCR-SSCP及生物信息学软件对10个错义突变SNPs的准确性进行验证,利用SAS 8.1的GLM程序分析其对新疆巩乃斯种羊场育种核心群300只细毛羊的遗传效应及与被毛纤维直径的关联性。结果表明,20个SNPs中10个是同义突变SNPs;10个错义突变SNPs验证后7个发生错义突变,导致蛋白性质改变,3个呈假阳性。突变后LAMB1蛋白疏水性更强,预测是不可溶性蛋白。SNP5中TT基因型个体纤维直径显著高于TC和CC基因型个体（P<0.05）,SNP9中GG和TT基因型个体纤维直径显著高于GT基因型个体（P<0.05）。虽然DNA混池全基因组重测序技术完整地检测到基因的SNPs,并将假阳性最小化,但还需要对结果进行验证。研究揭示LAMB1基因外显子多态性丰富,突变SNPs在外显子中分布不平衡,LAMB1基因SNP5（rs159769941）和SNP9（rs159769901）可以考虑作为影响细毛羊被毛纤维直径的有效遗传标记。     

从江香猪SIM1基因SNPs筛查及生物信息学分析

本研究以从江香猪为研究对象,二元杂交猪(从江香猪×野猪)、外三元杂交猪(杜×长×大)为对照,采用DNA池和直接测序技术对3个群体的SIM1基因7个外显子、部分内含子以及3'非翻译区序列进行多态性检测;利用生物信息学软件预测多态位点对SIM1基因mRNA二级结构和蛋白质一级、二级结构的影响。结果表明,在3个群体的SIM1基因中筛查到12个SNPs,C77T位于第5外显子,T29186C、A29195C位于第9内含子,C63T、C225T位于第10外显子,C107T、A426G、T583C、A586C、A605C、A615C位于第11外显子,G267T位于3'非翻译区。其中C77T、T29186C、A29195C、C63T、C225T、C107T、G267T为同义突变,A426G、T583C、A586C、A605C、A615C为错义突变;5个错义突变分别导致异亮氨酸(Ile)变为缬氨酸(Val)、亮氨酸(Leu)变为脯氨酸(Pro)、谷氨酸(Glu)变为丙氨酸(Ala)、谷氨酸(Glu)变为天冬氨酸(Asp)、天冬酰胺(Asn)变为组氨酸(His);根据在线软件预测,突变前后的SIM1基因mRNA二级结构和蛋白质一级、二级结构均会发生改变。     

中国荷斯坦牛TLR_2基因SNPs快速筛查及...等位基因频率估算

为了探讨Toll样受体2基因与荷斯坦牛乳腺炎抗性的相关性,本研究选取中国荷斯坦牛为试验对象构建DNA池,设计4对特异性引物,并结合克隆测序法对中国荷斯坦牛TLR_2基因编码区进行多态性筛查。结果显示,荷斯坦牛TLR_2基因编码区有6个核苷酸突变位点,分别为T602A-Intron2、G10900C-Exon2、G11047C-Exon2、A11076T-Exon2、C12077T-Exon2、T10634G-Exon2,其中T10634G-Exon2为错义突变,其余均为同义突变,SNPs突变前后等位基因频率有明显差异。6个SNPs等位基因频率分别由0.8067、0.7843、0.7959、0.7647、0.6897、0.8413突变为0.1933、0.2157、0.2041、0.2353、0.3103、0.1587。研究结果可为中国荷斯坦牛的分子抗病育种提供理论依据。     

Polymorphism of LAMB1 Gene and Its Association Analysis with the Fiber Diameter in Fine-wool Sheep

This study aimed to research the single nucleotide polymorphism (SNPs) of LAMB1 gene exon and its correlation with the fiber diameter in Fine-wool sheep.Based on DNA pools with the Re-sequencing technology to gain SNPs data,totally screened 20 SNPs of LAMB1 gene exon regions,at the same time,combining with direct sequencing method,PCR-SSCP and bioinformatics software to verify the accuracy of 10 missense mutations.The genetic effects of LAMB1 on fiber diameter at Xinjiang Kunes farm were analyzed by the GLM of SAS,totally 300 sheep.The results showed that 20 SNPs of LAMB1 gene were screened,which included 10 synonymous mutation SNPs and 10 non-synonymous mutation SNPs,after the validation,there were 7 missense mutation SNPs which led to the nature of protein change,3 SNPs were not mutated.LAMB1 protein became increasingly hydrophobic after mutating,predicting that was insoluble protein.TT genotype in the fiber diameter of the individual value was significantly higher than TC and CC genotypes on SNP5(P<0.05),GG and TT genotypes in the fiber diameter of the individual value was significantly higher than GT genotype on SNP9 (P<0.05).Although,all of the SNPs were completely detected by DNA pool with the Re-sequencing technology and minimized the false positives,it still need to be validated by direct sequencing.The results revealed that LAMB1 gene existed highly genetic diversity,mutated SNPs were unevenly distributed in exons,SNP5(rs159769941) and SNP9(rs159769901) could be considered as an effective genetic markers of Fine-wool sheep on fiber diameter.     

DNA池快速筛查奶牛TLR_2基因多态性及等位基因频率估算

为了探讨Toll样受体2基因多态性,本试验选取中国荷斯坦牛为试验对象构建DNA池,并结合直接测序法对TLR_2基因编码区进行多态性筛查。结果显示,快速筛查到6个核苷酸位点,分别为T~(602)A、G~(10900)C、G~(11047)C、A~(11076)T、C~(12077)T、T~(10634)G,其中T~(10634)G为错义突变,氨基酸由原来的谷氨酸(Gly)转变为天冬氨酸(Asp),SNPs突变前后等位基因频率有明显差异。研究结果可为中国荷斯坦牛的分子遗传育种提供理论依据。     

Practical capability of a DNA pool‐based genome‐wide association study using BovineSNP50 array in a cattle population

Genome‐wide association mapping for complex traits in cattle populations is a powerful, but expensive, selection tool. The DNA pooling technique can potentially reduce the cost of genome‐wide association studies. However, in DNA pooling design, the additional variance generated by pooling‐specific errors must be taken into account. Therefore, this study aimed to investigate factors such as: (i) the accuracy of allele frequency estimation; (ii) the magnitude of errors in pooling construction and in the array; and (iii) the effect of the number of replicate arrays on P‐values estimated by a genome‐wide association study. Results showed that the Illumina correction method is the most effective method to correct the allele frequency estimation; pooling errors, especially array variance, should be taken into account in DNA pooling design; and the risk of a type I error can be reduced by using at least two replicate arrays. These results indicate the practical capability and cost‐effectiveness of pool‐based genome‐wide association studies using the BovineSNP50 array in a cattle population.     

Allelic frequencies and association with carcass traits of six genes in local subpopulations of Japanese Black cattle

Marker‐assisted selection (MAS) is expected to accelerate the genetic improvement of Japanese Black cattle. However, verification of the effects of the genes for MAS in different subpopulations is required prior to the application of MAS. In this study, we investigated the allelic frequencies and genotypic effects for carcass traits of six genes, which can be used in MAS, in eight local subpopulations. These genes are SCD, FASN and SREBP1, which are associated with the fatty acid composition of meat, and NCAPG, MC1R and F11, which are associated with carcass weight, coat color and blood coagulation abnormality, respectively. The frequencies of desirable alleles of SCD and FASN were relatively high and that of NCAPG was relatively low, and NCAPG was significantly associated with several carcass traits, including carcass weight. The proportions of genotypic variance explained by NCAPG to phenotypic variance were 4.83 for carcass weight. We thus confirmed that NCAPG is a useful marker for selection of carcass traits in these subpopulations. In addition, we found that the desirable alleles of six genes showed no negative effects on carcass traits. Therefore, selection using these genes to improve target traits should not have negative impacts on carcass traits.     

8个牛品种的大理石花纹评分相关基因变异的微卫星DNA池分析

本研究以8个牛品种为研究对象,利用与大理石花纹评分基因相关的7个微卫星位点结合DNA池分析技术,探讨微卫星DNA多态性与8个牛品种大理石花纹评分间的关系,并根据性状同质性原理预测品种组合,以加快生产出高端"雪花"牛肉。结果表明:日本和牛与荷斯坦牛、安格斯牛、渤海黑牛的相似性系数均超过0.8,而日本和牛与其他牛品种的的相似性系数均小于0.8。利用MEGA4软件采用邻接法进行聚类,利木赞牛与西门塔尔牛先聚合在一起,再与草原红牛聚合;渤海黑牛与鲁西黄牛2个地方良种聚合在一起,以上5个品种聚为第1大类。荷斯坦牛与日本和牛聚合,再与安格斯牛聚在一起,它们聚为第2大类。根据本研究结果和国外肉牛杂交生产实践,从牛肉大理石花纹性状的同质性出发,建议利用日本和牛、荷斯坦牛为亲本,杂交生产高档"雪花"牛肉,以解决我国的肉牛牛源短缺问题。     

马传染性贫血病毒第19、26代驴胎皮肤细胞弱毒前病毒DNA全基因序列分析

不同代次马传染性贫血驴胎皮肤细胞弱毒(Fetal donkey dermal virus,FDDV)的免疫保护效果各不相同,只有第10～15代驴胎皮肤细胞弱毒具有良好的免疫保护效果,可作为疫苗使用,继续传代则疫苗的保护率下降。为确定有、无免疫保护效果的FDDV在基因水平上的差异,本实验对无免疫保护效果的第19、26代驴胎皮肤细胞弱毒前病毒DNA进行了全基因序列测定,并与已测序的疫苗毒株进行序列比较。第19代和第26代FDDV全基因核苷酸序列同源性高达99.5%,与疫苗毒株全基因核苷酸序列的同源性分别为96.9%、96.7%。LTR是EIAV在细胞传代中变异最显著的区域,第19、26代FDDV的LTR与疫苗毒的LTR同源性仅为89.6%、89.3%。马传贫病毒的gag基因高度保守,第19、26代FDDV与疫苗毒株的gag基因推导氨基酸序列仅有2个氨基酸不同。第19、26代FDDV与疫苗毒株的pol基因、env基因的推导氨基酸序列的同源性分别为98.9%、98.8%、93.7%、93.6%。由序列比较结果可以推断,第19代、第26代FDDV不具有免疫保护效果的主要原因可能是由于LTR和env基因的变异,导致病毒复制能力下降或免疫原性丧失,不能诱导机体产生良好的免疫反应。     

Molecular Characterization by LSSP‐PCR and DNA Sequencing of a Pathogenic Isolate of Leptospira interrogans from Brazil

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31–21%), Hardjo Norma (46–40%), Hardjo hardjobovis (18–10%), Mini (8–4%) and Wolffi (7–4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three‐dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single‐specific primer polymerase chain reaction (LSSP‐PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.